UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Phytoremediation and metal speciation in highway soils Padmavathiamma, Prabha Kumari 2010

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
24-ubc_2010_spring_padmavathiamma_prabha.pdf [ 15.44MB ]
Metadata
JSON: 24-1.0069527.json
JSON-LD: 24-1.0069527-ld.json
RDF/XML (Pretty): 24-1.0069527-rdf.xml
RDF/JSON: 24-1.0069527-rdf.json
Turtle: 24-1.0069527-turtle.txt
N-Triples: 24-1.0069527-rdf-ntriples.txt
Original Record: 24-1.0069527-source.json
Full Text
24-1.0069527-fulltext.txt
Citation
24-1.0069527.ris

Full Text

PHYTOREMEDIATION AND METAL SPECIATION IN HIGHWAY SOILS  by   Prabha Kumari Padmavathiamma     A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF     DOCTOR OF PHILOSOPHY   in  The Faculty of Graduate Studies  (Soil Science)     THE UNIVERSITY OF BRITISH COLUMBIA  (VANCOUVER)      April 2010    © Prabha Kumari Padmavathiamma, 2010  ii ABSTRACT  Research was conducted to develop a cost effective and environmentally friendly technology to limit the dispersal of metal contaminants from highway traffic in the soil to the surrounding natural environment. The study comprised preliminary field measurements followed by two pot experiments and a field study. The first study evaluated the phytoextraction/ phytostabilisation potential of five plant species: Brassica napus L (rape), Helianthus annuus L. (sunflower), Lolium perenne L (perennial rye grass), Poa pratensis L (Kentucky blue grass) and Festuca rubra L (creeping red fescue) for metals (Cu, Mn, Pb and Zn), in soils with different metal contamination levels. The promising plant species identified were Lolium perenne, Festuca rubra and Poa pratensis. Total soil and plant metal concentrations, as well as the relative metal partitioning in different soil fractions and in plants were determined to provide an estimate of the mobility and potential bioavailability of metals in the soil. The second study evaluated the effectiveness of soil-plant-amendment interaction in immobilising metals in the soil. The amendments included lime, phosphate and compost individually and in combination, and were applied to the plant species: Lolium, Poa and Festuca. Maximum metal immobilisation was achieved in the soil by the combined application of amendments in conjunction with growth of Festuca for Cu, Poa for Pb and Zn and Lolium for Mn. The results obtained from first and second studies were confirmed by conducting field studies. A completely randomized factorial experiment in split plot design with three plant species (Lolium, Poa, and Festuca) individually and in combination, with and without soil amendments was conducted along Highway 17 soil in southwest British Columbia. The influence of root-soil interactions and seasonal influence on the solubility and bioavailability of metals in the soil with and without soil amendments was also evaluated. The best management practices (BMP) developed from the study have the applicability for phytostabilisation of metal contaminated sites and can be suggested as a risk management activity, reducing long-term associated risks.     iii TABLE OF CONTENTS  ABSTRACT .......................................................................................................................ii TABLE OF CONTENTS .................................................................................................iii LIST OF TABLES..........................................................................................................viii LIST OF FIGURES...........................................................................................................x LIST OF ABBREVIATIONS........................................................................................xiii ACKNOWLEDGEMENTS...........................................................................................xiv STATEMENT OF COAUTHORSHIP .........................................................................xv  1 INTRODUCTION....................................................................................................... 1   1.1 Statement of the problem...................................................................................... 1  1.2  Scope and objectives............................................................................................. 4  1.3  Research plan ........................................................................................................ 5   1.3.1. Preliminary investigations.............................................................................. 5   1.3.2.  Identifying genera of plants which could be suitable for               B.C. climatic conditions ................................................................................. 5   1.3.3.  Effect of soil amendments in influencing the plants to immobilize  metals in the soil............................................................................................. 6  1.3.4.  Field experiments for phytostabilisation at highway site ............................... 7  1.4 Research contributions ......................................................................................... 7  1.5  Dissertation organization...................................................................................... 8  1.6  References .......................................................................................................... 10  2 PHYTOREMEDIATION TECHNOLOGY: HYPER-ACCUMULATION       METALS IN PLANTS.............................................................................................. 13   2.1 Introduction......................................................................................................... 13  2.2  Categories of phytoremediation.......................................................................... 18       2.2.1 Phytostabilization ........................................................................................ 19       2.2.2  Phytofiltration............................................................................................... 24   2.2.3  Phytovolatilization........................................................................................ 27   2.2.4  Phytoextraction............................................................................................. 29   2.2.4.1 Types of phytoextraction ....................................................................... 33        2.2.4.2  Successful factors for phytoextraction of heavy metals ........................ 33  2.3. Handling of hazardous plant biomass after phytoremediation ........................... 38  iv  2.4.  Conclusions......................................................................................................... 39  2.5  References........................................................................................................... 40  3. PRELIMINARY EXAMINATION OF FIELD MEASUREMENTS OF METAL ACCUMULATION - HEAVY METAL STATUS OF SOILS AND PLANTS PRIOR TO PHYTOREMEDIATION ALONG HIGHWAYS ............ 53   3.1 Introduction......................................................................................................... 53  3.2  Materials and methods ........................................................................................ 54   3.2.1 Site description ............................................................................................. 54   3.2.1.1 Trans-Canada Highway & 176 th St. Site description............................. 54        3.2.1.2 Highway 17 site ..................................................................................... 55   3.2.1.3 Background locations ............................................................................ 55   3.2.2 Collection of samples and laboratoratory analysis....................................... 56  3.3  Results and discussion ........................................................................................ 57  3.4  Conclusions......................................................................................................... 61  3.5  References........................................................................................................... 62  4. PHYTOREMEDIATION OF METAL-CONTAMINATED SOIL IN       TEMPERATE HUMID REGIONS OF BRITISH COLUMBIA, CANADA ...... 63   4.1 Introduction......................................................................................................... 63  4.2  Materials and methods ........................................................................................ 65   4.2.1  Experimental details ..................................................................................... 65       4.2.2  Bio-metric observations ............................................................................... 67       4.2.3  Laboratory analysis ...................................................................................... 67    4.2.4  Statistical analyses........................................................................................ 67  4.3.  Results and discussion ........................................................................................ 68   4.3.1  Metal concentrations in plants...................................................................... 69       4.3.2  Metal content in plants ................................................................................. 71   4.3.3  Metal accumulation characteristics .............................................................. 74   4.3.4  Relationships of metal concentration and bio-metric characters of plants... 76   4.3.5  Relationships of metal content and biomass of plants ................................. 78  4.4 Conclusions and recommendations..................................................................... 80  4.5  References........................................................................................................... 82  4. EXPLORATION OF PHYTOREMEDIATION AND ITS EFFECT ON MOBILITY OF METALS IN SOIL – A FRACTIONATIONSTUDY................ 86   5.1 Introduction......................................................................................................... 86  v  5.2  Materials and methods ........................................................................................ 88  5.3  Results and discussion ........................................................................................ 90   5.3.1 Physico-chemical properties of soil as influenced by plant growth............. 91   5.3.2  Metal fractionation in Soils .......................................................................... 93   5.3.3 Metal comparison ......................................................................................... 97       5.3.4  Relationship between soil pH and metal fractions ....................................... 97       5.3.5  Relationship of plant metal concentrations to soil metal fractions .............. 99  5.4 Conclusions and recommendations................................................................... 103  5.5  References......................................................................................................... 104  6 EFFECT OF AMENDMENTS ON PHYTOAVAILABILITY AND FRACTIONATION OF COPPER AND ZINC IN CONTAMINATED  SOIL ........................................................................................................................ 108   6.1  Introduction....................................................................................................... 108  6.2  Materials and methods ...................................................................................... 110  6.3  Results and discussion ...................................................................................... 112   6.3.1  Effect of soil amendments on metal concentrations and                      uptake in plants........................................................................................... 112       6.3.2  Accumulation of Cu and Zn in plants ........................................................ 116       6.3.3  Metal fractionation in the soil .................................................................... 119       6.3.4  Relationship between soil pH and metal fractions ..................................... 122       6.3.5  Relationship of plant metal concentration to soil metal fractions .............. 124       6.3.6  Plant metal concentrations and biometric characteristics........................... 125  6.4  Conclusions and recommendations................................................................... 126  6.5  References......................................................................................................... 128  7. PHYTOAVAILABILITY AND FRACTIONATION OF LEAD AND MANGANESE IN CONTAMINATED SOIL FOLLOWING APPLICATION OF THREE AMENDMENTS ................................................................................ 132   7.1 Introduction....................................................................................................... 132  7.2  Materials and methods ...................................................................................... 133  7.3  Results and discussion ...................................................................................... 135       7.3.1 Metal concentrations and uptake in plants ................................................. 135       7.3.2  Accumulation characteristics of Pb and Mn in plants................................ 137       7.3.3  Pb and Mn fractions in the soils ................................................................. 139   7.3.4  Relationships between soil pH and soil metal fractions............................. 143   vi    7.3.5 Relationships between soil metal concentrations and the    Enrichment Coefficient .............................................................................. 144       7.3.6  Relationships between soil metal fractions and plant                       metal concentrations.................................................................................. 145   7.3.7  Relationships between soil properties and metal uptake by plants ............ 146  7.4 Conclusions....................................................................................................... 149  7.5  References......................................................................................................... 151  8. RHIZOSPHERE INFLUENCE AND SEASONAL IMPACT       ON PHYTOSTABILISATION OF METALS – A FIELD STUDY ................... 154   8.1 Introduction....................................................................................................... 154  8.2  Materials and methods ...................................................................................... 155       8.2.1 Experiment details ...................................................................................... 155       8.2.2  Collection of samples and laboratory analysis ........................................... 156   8.2.3  Statistical analysis ...................................................................................... 157  8.3 Results and discussion ...................................................................................... 157       8.3.1 pH and Electrical Conductivity .................................................................. 158      8.3.2  Metal concentrations in soil ....................................................................... 159       8.3.3  Metal fractionation in soil .......................................................................... 160       8.3.4  Metal concentrations and uptake in plants ................................................. 166       8.3.5  Metal accumulation charecteristics in plants.............................................. 171       8.3.6  Bulk soil vs Rhizosphere soil ..................................................................... 174        8.3.6.1 pH ........................................................................................................ 174   8.3.6.2 Metal Fractionation in RS and BS....................................................... 175        8.3.6.3 Total metal concentrations in RS and BS............................................ 178  8.4 Conclusions....................................................................................................... 180  8.5  References......................................................................................................... 181  9. CONCLUSIONS AND RECOMMENDATIONS ................................................ 186   9.1 Conclusions....................................................................................................... 186    9.2.  Recommendations and future work .................................................................. 189  9.3. References......................................................................................................... 192   APPENDICES ......................................................................................................... 193   Appendix A - Preliminary studies ............................................................................. 193  Appendix B - Stage I study ....................................................................................... 199  vii  Appendix C - Stage II study ...................................................................................... 201  Appendix D - Stage III study .................................................................................... 206       Appendix E - QA/QC. Abstract of ANOVA – Field Experiment............................. 211       Appendix F - List of publications from thesis .......................................................... 220                                viii  LIST OF TABLES  Table 2.1 Cost of different remediation technologies .................................................. 15 Table 2.2  Different mechanisms of phytoremediation ................................................. 18 Table 2.3  Approaches to revegetation .......................................................................... 20 Table 2.4  Summary of research results – Phytostabilisation........................................ 22 Table 2.5  Summary of research results – Phytofiltration ............................................. 26 Table 2.6  Effect of typical levels for heavy metals in plants ....................................... 30 Table 2.7(a) Examples of hyperaccumulators and their bioaccumulation potential......... 30 Table 2.7(b) Hyperaccumulators and their bioaccumulation potential ............................. 31 Table 2.8  Recent reports on phytoextraction................................................................ 36 Table 3.1  Basic soil characteristics of Highway soils .................................................. 57 Table 3.2  Soil metal concentrations with distance from the highway (HW1) ............. 58 Table 3.3  Metal concentrations in back ground (BG) sites .......................................... 58 Table 3.4  Root/ Shoot ratio, Enrichment Coefficient and Translocation Factor.......... 61 Table 4.1  Metal concentrations according to British Columbia CSR  (Contaminated Sites Regulation) standards and studied soil  metal concentrations in the pot study ........................................................... 65 Table 4.2  Key characteristics of the original soil sample............................................. 68 Table 4.3  Metal uptake (µg/pot) by plants (roots, shoots and total) at 90 and 120 DAS ....................................................................................................... 72 Table 4.4  Correlations between metal concentrations (mg/kg) in plants  (shoot) at 120 DAS....................................................................................... 73 Table 4.5  Translocation Factor (TF) and Enrichment Coefficient (EC) of metals ...... 75 Table 4.6  Correlations between metal concentrations (mg/kg) in plants  and biometric characters at 120 DAS........................................................... 77 Table 5.1  Experimental Program for identification of plant species for  Phytostabilisation ......................................................................................... 89 Table 6.1  Experimental Program for soil-plant-amendment interaction.................... 110 Table 6.2  Enrichment Coefficient (EC) and Translocation Factor (TF) in different  treatments by plant  growth and amendment addition ............................... 117 Table 6.3  % partitioning of Cu and Zn in soils with and without plant growth ......... 120 Table 6.4  Correlation coefficients between soil pH and metal fraction ..................... 124  ix Table 6.5  Correlations between plant (root and shoot) metal concentrations  and soil metal fractions............................................................................... 125 Table 6.6.  Correlation coefficients between plant biometric characters and  metal concentrations................................................................................... 126 Table 7.1  Physico chemical properties of the studied soils........................................ 135 Table 7.2  Metal concentrations and metal uptake by the plants (root and shoot) ...... 136 Table 7.3  ECroot, ECshoot and TF for Pb and Mn ......................................................... 138 Table 7.4  % partitioning of Pb and Mn in soils with and without plant growth ........ 140 Table 7.5  Relationship between Enrichment Coefficient (EC) and total soil  metal concentration (mg/kg) ...................................................................... 144 Table 8.1  Experimental program for field study ........................................................ 156 Table 8.2  Key soil characteristics before the field experiment .................................. 157 Table 8.3  % metal fractionation in the soil before plant growth ................................ 160 Table 8.4  Metal concentrations in plants (mg/kg)...................................................... 167 Table 8.5  Enrichment Coefficient (ECroot and ECshoot) and Translocation  Factor (TF) of metals with and without amendments ................................ 172     x LIST OF FIGURES  Figure 2.1 Heavy metal content of road-side soils from  (a) Brussels-Ortend, Belgium (Albasel and Cottenie, 1985);  (b) Osogobo, Nigeria (Fakayode and Olu-Owolabi, 2003);  (c) West bank, Palestine (Swaileh et al., 2004);  (d) A31 between Nancy and France (Viard et al., 2004) ............................ 14 Figure 2.2 Heavy metal content in plants growing on contaminated sites                       (Yoon et al., 2006).                       (a) Bahia grass (Paspalum notatum);                       (b) Wire grass (Gentiana pennelliana);                       (c) Ticktrefoil (Desmodium paniculatum);                       (d) Flats edge (Cyperus esculentus);                       (e) Bermuda grass (Cynodon dactylon)....................................................... 17 Figure 2.3  Schematic mechanism of phytostabilization............................................... 19 Figure 2.4  Schematic mechanism of phytovolatilization ............................................. 27 Figure 2.5  Schematic mechanism of phytoextraction .................................................. 29 Figure 3.1  Soil metal concentrations with distance from the highway (HW 17)   (mean values±SD, n = 3)............................................................................. 59 Figure 3.2  Concentration of metals in the plants that spontaneously colonised                        the study sites (mean values±SD, n = 3) .................................................... 60 Figure 3.3  Metal concentrations (mg/kg) in moss (Rhytidiadelphus squarrosus) ....... 61 Figure 4.1  Germination per cent (mean values). Error bars represent                        means ±S.D for three replicates ................................................................. 69 Figure 4.2  Metal concentrations in plants at 120 DAS.  (a) Cu, (b) Pb, (c) Mn, (d) Zn.  (1. LB0, 2. LBA, 3. FB0, 4. FBA, 5. HB0, 6. PB0, 7. PBA, 8. BrB0).  Error bars represent means ±S.D. for three replicat.................................... 70 Figure 4.3  Relationship between metal content in plants and plant  biomass (dry weight) at 120 DAS.  (a) Root biomass and Cu content in roots.  (b) Shoot biomass and Cu content in shoots.  (c) Root biomass and Pb content in roots.  (d) Shoot biomass and Pb content in shoots................................................ 79 Figure 5.1     (a) pH and (b) Electrical Conductivity of soils at 90 and 120 DAS.  Error bars represent ±S.D of means of three replicates.  F-values for pH and Electrical Conductivity are significant at P <0.05..... 92 Figure 5.2  Metal fractionation (%) in soil by the influence of plant growth at 90 and  120 DAS. n = 3, F-values are significant at P <0.05. LB0 (Lolium B0 soil),  LBA (Lolium BA soil), FB0 (Festuca B0 soil), FBA (Festuca BA soil),  PB0 (Poa B0 soil), PBA (Poa BA soil), HB0 (Helianthus B0 soil)                       and BrB0 (Brassica B0 soil) ........................................................................ 96  xi Figure 5.3 Effect of soil pH on soil metal fractions (exchangeable, oxide,  organic and residual) at 120 DAS.  (a) soil pH and Cu fractions, (b) soil pH and Pb fractions,  (c) soil pH and Mn fractions, (d) soil pH and Zn fractions......................... 98 Figure 5.4  Relationship between plant metal concentrations (in root and shoot)  and soil metal fractions ............................................................................. 100 Figure 6.1  Metal concentration in plants (in root and shoot).  (a) Cu concentrations in Lolium.  (b) Zn concentrations in Lolium,  (c) Cu concentrations in Festuca,  (d) Zn concentrations in Festuca,  (e) Cu concentrations in Poa,  (f) Zn concentrations in Poa.  BA - spiked soil, BAL - spiked soil plus lime,  BAP - spiked soil plus phosphate,  BAO - spiked soil plus compost,  BALPO - spiked soil plus lime, phosphate and compost. F significant                        at P<0.05 for both Cu and Zn................................................................... 113 Figure 6.2  Soil pH as influenced by plant growth and amendment application.   * - F significant at P<0.05. B0 – Initial soil, BA – Spiked soil,  BAL - Spiked soil plus lime, BAP - Spiked soil plus   phosphate,  BAO - Spiked soil plus compost, BALPO - Spiked soil plus lime,   phosphate and compost. ............................................................................. 114 Figure 6.3  Metal uptake by plants. BA - Spiked soil, BAL - Spiked soil plus lime,  BAP - Spiked soil plus phosphate, BAO - Spiked soil plus compost,  BALPO - Spiked soil plus lime, phosphate and compost  Mean values, n = 3. F significant at P<0.05.............................................. 116 Figure 6.4  Relationship between soil metal concentrations and Enrichment  Co-efficients (EC) for root and shoot........................................................ 118 Figure 6.5  Partitioning of Cu and Zn in soil by the effect of amendments and plants  BA - Spiked soil, BAL - Spiked soil plus lime,  BAP - Spiked soil plus phosphate,  BAO - Spiked soil plus compost,  BALPO - Spiked soil plus lime, phosphate and compost.  Mean values, n = 3. F significant at P<0.05.............................................. 121 Figure 6.6  Relationship between soil pH and metal fractions.  (a) soil pH and Cu fractions in Lolium soil,  (b) soil pH and Zn fractions in Lolium soil,  (c) soil pH and Cu fractions in Festuca soil,  (d) soil pH and Zn fractions in Festuca soil,  (e) soil pH and Cu fractions in Poa soil,  (f) soil pH and Zn fractions in Poa soil..................................................... 123 Figure 7.1  Mn and Pb fractionation in soils.  B0 - Initial soil, BA - Spiked soil,  BAL - Spiked soil plus lime,  BAP - Spiked soil plus phosphate,  BAO - Spiked soil plus compost,  BALPO - Spiked soil plus lime, phosphate and compost ......................... 141   xii Figure 7.2  Relationship between soil pH and metal fractions in soil  (a) soil pH and Pb fractions in the soil.  (b) soil pH and Mn fractions in the soil .................................................... 143 Figure 7.3  Relationship between plant metals and soil metal fractions.  (a) Exchangeable Pb and Root Pb,  (b) Oxide Pb and Root Pb,  (c) Exchangeable Mn and Root Mn,  (d) Exchangeable Mn and Shoot Mn,  (e) Oxide Mn and Root Mn,  (f) Oxide Mn and Shoot Mn...................................................................... 145 Figure 7.4 Relationship between soil properties and metal uptake by the plants.  (a) Pb uptake and soil pH,  (b) Mn uptake and soil pH,  (c) Pb uptake and % organic matter in the soil,  (d) Mn uptake and % organic matter in the soil,  (e) Pb uptake and available P in the soil,  (f) Mn uptake and available P in the soil .................................................. 147 Figure 8.1  Seasonal influences on pH and electrical conductivity of soil.  T0 – without amendments, T1 – with amendments (lime plus phosphate) ................................................................................ 159 Figure 8.2 % metal fractionation in soil by the combined influence of plants,  amendments and seasons. (a) – Cu, (b) – Pb, (c) – Mn, (d) – Zn.  T0 – without amendments, T1 – with amendments.  L – Lolium, F- Festuca, P – Poa,  C – Combination (Lolium + Festuca + Poa).  FS – Fallow soil. Mean values, n = 3.  F significant at P<0.05 for all the four metals .......................................... 162 Figure 8.3  Metal uptake by the plants during three seasons.  (a) – Cu uptake, (b) – Pb uptake, (c) – Mn uptake,  (d) – Zn uptake. Mean values. n = 3,  F significant at P <0.05. L- Lolium, F – Festuca,  P – Poa, C- Combination (.Lolium + Festuca + Poa).  S – summer, A – autumn, W – winter.  T0 – without amendments, T1 – with amendments .................................. 169 Figure 8.4 ECR (Enrichment Coefficientroot), ECS (Enrichment Coefficientshoot),  TF (Translocation Factor) of metals during different seasons.                        Mean values, n = 3. F significant at P<0.05 .for all                         the four metals ......................................................................................... 173 Figure 8.5  pH of Rhizosphere soil and Bulk soil. L – Lolium, F – Festuca, P- Poa,  C - Combination. Mean values, n = 3. F significant at P<0.05 ................ 174 Figure 8.6  Metal fractions in Rhizosphere soil and Bulk soil. (a) Cu, (b) Pb,                      (c) Mn, (d) Zn. RS – Rhizosphere soil, BS – Bulk soil.                      T0 – without amendments, T1 – with amendments.                      Mean values, n = 3. F significant at P<0.05 .............................................. 176 Figure 8.7  Total metal concentrations in Bulk soil and Rhizosphere soil.                      RS – Rhizosphere soil, BS – Bulk soil. Mean values, n = 3. F significant                      at P<0.05 for all the four metals. ............................................................... 178    xiii LIST OF ABBREVIATIONS B0 - Original soil BA - Spiked soil BAL - BA soil + lime BAP - BA soil + phosphate BAO - BA soil + compost BALPO - BA soil + lime + phosphate + compost BMP- Best Management Practices BG - Background BrB0 - Brassica in B0 soil BS - Bulk soil CEC - Cation exchange capacity CSR - Contaminated Sites Regulations DAS - Days after sowing EC - Enrichment Coefficient ECR - Enrichment Coefficient (Root) ECS - Enrichment Coefficient (Shoot) EPA - Environmental Protection Agency FB0 - Festuca in B0 soil FBA - Festuca in BA soil HB0 - Helianthus in B0 soil HW - Highway LB0 - Lolium in B0 soil LBA - Lolium in BA soil MMT - Methyl cyclopentadienyl manganese tricarbonyl PB0 - Poa in B0 soil PBA - Poa in BA soil RD – Relative difference RS - Rhizosphere soil SD - Standard deviation SE – Standard error TEL - Tetraethyl Lead TCH - Trans Canada Highway TF - Translocation Factor  xiv ACKNOWLEDGEMENTS  I express my sincere gratitude and appreciation to Dr. Loretta Li, my supervisor, for accepting me as her student and for her excellent guidance, patience, friendship and support during the course of my studies. I value her wisdom, advice and encouragement.  This research project would not have been possible without the support and help of Dr. Les Lavkulich, member of my supervisory committee. I am deeply indebted to him for his guidance, help and support during the course of this research and my entire Ph.D program. Our conversations and his expert advice have shaped and motivated my academic and professional growth.  My sincere thanks and heartfelt gratitude to Dr. Art Bomke, member of my supervisory committee, for his valuable input in designing experiments, presentation of data and help throughout my learning journey.  I am also deeply grateful to Dr. Peter Jolliffe, member of my supervisory committee for his help, encouragement and critical review of manuscripts.  I extend my appreciation to Dr. Tony Kozak for providing necessary help in the statistical analysis of the data.  I appreciate the assistance and help I received from Dr. Brent A. Hine, Will and Nicci during the course of this study. I also thank all my colleagues from Soil Science and Civil Engineering for their encouragement and friendship.  My special gratitude to my husband Kishore and my son Varun for their unconditional understanding, patience, help and emotional support during the course of this study.  I also convey my profound gratitude to NSERC (Natural Sciences and Engineering Research Council of Canada) and the B.C Ministry of Transportation and Infrastructure for providing financial support.  xv  STATEMENT OF COAUTHORSHIP  Results derived from the research of this thesis form the basis of six publications in peer- reviewed journals and seven papers in refereed conference proceedings.  Formulation of the project proposal, research work involving sample collection, conducting pot experiments, laying out of field experiment, recording phenotypical traits, analysis of soil and plant samples, conducting instrumental analyses using Varian Spectre AA 220 Multi-element Fast Sequential Atomic Absorption Spectrometer, tabulation of data, statistical analysis of data and preparation of manuscripts were performed by Prabha Kumari Padmavathiamma.  Dr. Loretta Li as supervisor helped to develop the research program, reviewed the progress of my work and critically reviewed the manuscripts. 1. INTRODUCTION  1.1 Statement of the problem  Metal contamination as a result of human activity is of major concern for ecosystem and human health. A common pathway for metal contamination is through the atmosphere and subsequent deposition onto soil or water. Once the metal contaminants reach the soil or water, physico- chemical processes govern the reactions and fate of these toxicants. Transportation systems, notably highways, are a universal concern for their contamination of adjacent right-of-ways. Highway systems transect all forms of landscapes in a diverse array of environments. The fate of transport of related metals along highways has received considerable research attention, but the development of cost-effective, non-destructive remediation techniques needs further consideration. This study focuses on two highway sites in southwestern British Columbia and assesses the levels of contamination arising from highway traffic, the use of commercially available plants for remediation and the dynamics of metal uptake and reactions under a range of seasonal conditions.  Soil pollution by metals differs from air or water pollution, because metals persist much longer in soils than in other compartments of the biosphere (Asami, 1984; Alkorta et al., 2004). Also certain metals such as Pb and Cr may not be removable and reside virtually permanently (Kabata-Pendias and Adriano, 1995). Since the residence time of metals in soil is of the order of thousands of years (McGrath, 1987), novel technological approaches are required to remediate excess toxic metals.  Apart from vehicular traffic, other sources of metal contaminants in soils include metalliferous mining and smelting sites, metallurgical industries, sewage sludge applications, warfare and military training areas or shooting ranges, waste disposal sites, agricultural fertilizers and electronic industries (Alloway, 1995). Road sediments typically contain elevated levels of metals, which may be mobilized by runoff waters (Sansalone and Buchberger, 1997; Sezgin et al., 2003). Heavy metal contaminants in road sediments are derived from: engine and brake pad wear, (e.g. Cd, Cu, and Ni) (Viklander, 1998; Varrica et al., 2003); lubricants (e.g. Cd, Cu and Zn) (Birch and Scollen, 2003); exhaust emissions, (e.g. Pb and Mn) (Al-Chalabi and Hawker, 2000; Sutherland et al., 2003; Zayed et al., 2003); and tire abrasion (e.g. Zn) (Smolders and  2 Degryse, 2002). The availability of metals to plants as well as lower food-chain organisms along roadside corridors is a potential concern (Turner et al., 2001). Although the use of leaded gasoline has been suspended in North America and most of the industrialized world, lead continues to be used in approximately 74 countries around the globe, predominantly in sub- Saharan Africa, the Middle East and most of Asia (Hodes et al., 2003). The use of tetraethyl lead (TEL) as an antiknock compound for gasoline engines until the early 1980s and its subsequent replacement by methyl cyclopentadienyl manganese tricarbonyl (MMT) have led to considerable emissions of Pb and Mn to the environment.  Metal contaminants from all these sources accumulate in soils to levels which can cause ecological and human health risks. The behaviour of metals (distribution, chemical forms, bio- availability and micro environmental effects) can be related to various pedogenic features such as soil pH, electrical conductivity, redox potential, organic matter content, cation exchange capacity and other surface properties. These features interact and result in certain processes (e.g. accumulation, removal, translocation and transformations) that operate in the soil over time, leading to differential distribution of metals in various fractions. The distribution of metal fractions govern the mobility/immobility of metals, controlling the off-site migration of the soluble/mobile fraction either to surface water or ground water, where they contaminate drinking water resources and enter the food chain (Adriano et al., 2004). Prolonged and sub-clinical exposure to metals causes various health problems in plants, animals and human beings, including disorders such as cancer, neurological and psychiatric disorders, Parkinson’s disease and kidney failure (Baudouin et al., 2002).  Given the high costs associated with conventional (ex-situ) cleanup methods and the large (and growing) number of metal-contaminated sites in Canada, there is considerable interest in developing innovative protocols such as phytoremediation strategies, adapted to the extreme climatic conditions in Canada (Environment Canada, 2003). Phytoremediation involves the use of plants to remove, transfer, stabilize and/or degrade contaminants in soil, sediment and water (Hughes et al. 1997). It is a continuum of various strategies that include both treatment and removal and has the potential to remediate metal contaminated sites, while maintaining the functional and ecological integrity of soil after remediation (Chaney et al.1997, Baker and Brooks 1989; Wang et al, 2006). Phytostabilisation is a feasible and practical remediation strategy for busy contaminated sites. There is no attempt to extract the metals from soil, but to  3 immobilise them. In contrast, phytoextraction removes metals from the soil, but, the disposal of metal-loaded plants is expensive and can lead to risk enhancement. Reducing the environmental impact by holding the metal-pollutants at the source location in non-mobile forms so that they do not interfere with the normal life processes of the vegetated cover is phytostabilisation (Smith and Bradshaw, 1979). Metal mobility and bioavailability can be reduced by growing selected plants or adding various immobilizing agents to the soil, or a combination of both (Smith and Bradshaw, 1979; Simon, 2005, Mench et al., 2000). Therefore the selection of suitable plant species and metal specific soil amendments should be geared towards the maximum partitioning of metals into the immobile fractions in soil. Many published reports are available on the suitabilities of different plant species for phytoextraction and phytostabilisation (Clemente et al., 2006; Conesa et al., 2006; Zhu et al., 2007 etc.) or use of amendments in metal stabilisation (Kumpiene et al., 2007; Simon, 2005; Mench et al., 2000 etc.). Most of the reported studies employed hydroponics, focusing on a single metal and assessing the remediation potential only at one stage of growth (Hamlin and Parker, 2006; Weng et al., 2005; Meyers et al., 2008). Also, there is a lack of research addressing the phytoremediation of roadside soils subjected to multi- component metal solutions, like those subjected to continuous atmospheric and highway runoff loadings. The land associated with highways in BC constitutes about 12,000 km of roads that are approximately 30 m wide (right-of-way). About 1/3 of the highways are unpaved (comprise millions of kilometres around the world) and support plant and animal life (Precciado and Li, 2006). The challenge is to find economical and efficient methods to effectively limit the dispersal of these contaminants into surface water and ground water and to protect the surrounding natural environments.  The present study focuses on phytostabilisation of major metals, i.e. Cu, Pb, Mn, and Zn in highway soils using plants that require minimal harvest and other maintenance. Investigations were based on the chemical protocols for metal accumulation and fractionation in soil (total and selective sequential extraction for metals), entry of metals into the plants, the seasonal impacts on metal dynamics in soil and rhizosphere influence on soil metal chemistry. The study provided extensive information on the mobility/immobility of metals as influenced by plant growth at various stages of growth, accumulation characteristics and translocation properties of metals in different plant species, the use of amendments in complementing the plant effect on metal immobilisation, the influence of pedogenic features on metal mobility/immobility, the root soil interactions on metal dynamics in soil and the seasonal impact on metal speciation in the soil.  4 The study provided guidance towards determining the BMP (Best Management Practices) for stabilisation of metal contaminants (Cu, Pb, Mn and Zn), involving suitable plants and soil amendments and considering seasonal influences and root soil metal interactions in the actual field scenario.  1.2 Scope and objectives  The present study was undertaken with the objective of developing an enhanced phytoremediation technology to reduce metal contamination in soils along highways so as to reduce negative environmental impacts. The following tasks were undertaken in support of this objective:  a) Identifying genera of plants which could be suitable for BC climatic conditions and soils, and be effective for phytoremediation. Vigorously growing plants in polluted sites were collected and their efficiencies for phytoremediation were compared with known phytoremediating plant species, native to North America (Environment Canada’s database PHYTOREM).  b) Growing the identified plants in soils with different metal contamination levels to study the removal of heavy metals at different stages of plant-growth. Factors investigated for each crop included total removal of heavy metals, optimum growth time of the species, biomass production and root response.  c) Determining the effect of soil properties such as pH, organic matter content, electrical conductivity and cation exchange capacity in influencing the plants to immobilise metals with and without soil amendments.  d) Conducting field experiments on phytostabilisation at a highway site featuring plants and amendments identified as promising from the previous experiments.     5 1.3 Research plan  1.3.1. Preliminary investigations  Preliminary studies included characterization of the type and extent of contamination of the study site, identification of native plants and assessment of their metal accumulation characteristics. The concentration of metals (Cu, Pb, Mn and Zn) in the study site, the forms in which they were found, plants that spontaneously colonized the site and their metal partitioning were investigated. Soil samples were collected at horizontal distances of 1, 2, 4, 6, and 8 m from each  road side investigated (HW1 and HW17) at two depth intervals, 0-15 cm and 15-30 cm. Plants were collected during winter and summer and their metal accumulation characteristics and translocation properties were studied. Information from this study provided insight for formulating the research program for the subsequent studies. A research paper based on this work is presented in Chapter 3.  1.3.2. Identifying genera of plants which could be suitable for B.C climatic conditions and soils and be effective in phytoremediation  This Stage I study involved a systematic and comprehensive effort to assess the phytoremediation potential of five plant species, Lolium perenne L (perennial rye grass), Festuca rubra L (red fescue), Helianthus annuus L (sunflower), Poa pratensis L (Kentucky bluegrass) and Brassica napus L (rape), commonly available in regions with temperate maritime climate, for a highway soil in southwest British Columbia. These species were selected because of their known success in phytoremediation elsewhere and readily available seeds for remediation protocols. Comprehensive pot tests with completely randomized experimental design using five plant species and four metals commonly found in highway roadside soils (i.e. Cu, Pb, Mn and Zn) were carried out under outdoor conditions. The soil used for this research was collected from the back yard of Surrey Fire Hall No. 5, located 1 km north of the intersection of TCH (Trans Canada Highway) with the 176  Street overpass in Surrey, British Columbia. This site was selected since it is located away from major traffic corridor and has minimum anthropogenic disturbance. Soils with three different metal concentrations were studied: (a) B0,  the original soil containing 52 mg/kg Cu, 93 mg/kg Pb, 215 mg/kg Mn and 70 mg/kg Zn (b) BA,  the original soil spiked with addition of all four metals to give total Cu, Pb, Mn, and Zn concentrations of of 80, 146, 408 and 148 mg/kg, respectively. (c) BC, the original soil spiked to provide total Cu, Pb,  6 Mn, and Zn concentrations of 520, 1100, 2160, and 1600 mg/kg, respectively. The research protocol involved: (1) estimating the metal uptake by plants at different growth stages; (2) determining the translocation properties and metal accumulation characteristics of the studied plant species; (3) investigating the distribution of metal fractions in the rhizosphere at two different growth stages under variable multimetal contamination levels; (4) examining the relationship of metal fractions to physico-chemical properties of soil; and (5) assessing the efficiencies of these plants for phytoextraction and phytostabilisation in soils. This study helped to identify the best plant species for phytostabilisation under the climatic and soil conditions of southwest British Columbia. Research papers based on the results are presented in Chapters 4 and 5.  1.3.3. Effect of soil amendments in influencing plants to immobilize metals in soil  This Stage II study was conducted to compare the efficiencies of the selected plants (from Stage 1) for phytostabilisation in soil with and without soil amendments. Plant species Lolium perenne L, Festuca rubra L and Poa pratensis L were tested in the presence of three soil amendments (lime, phosphate and compost, both individually and in combination) to assess the effect of soil- plant-amendment interaction on phytostabilisation of Cu, Pb, Mn and Zn. The efficiency of treatments to stabilize metals was assessed on the basis of metal speciation in soil, partitioning of metals in plants, and metal uptake by the plants. The effects of soil properties such as pH, CEC, organic matter content and electrical conductivity in influencing the plants to immobilize metals with and without soil amendments were evaluated. The original soil collected from the backyard of Surrey Fire Hall No. 5, near the main highway intersection (HW 1 with 176 street in Surrey, British Columbia) was spiked with addition of all four metals to give total concentrations of Cu, Pb, Mn, and Zn of 80, 146, 408 and 148 mg/kg, respectively. To the spiked soils, amendments such as lime, phosphate and compost were added individually and in combination and plants grown. The study was conducted as a pot experiment in a completely randomized design (CRD) with 18 treatments and three replications. The experiment was performed in the greenhouse during the period, August 2006 to November 2006. Research papers based on these results appear in Chapters 6 and 7.     7 1.3.4. Field experiments for phytostabilisation at a highway site  The field study is the Stage III study, and it was undertaken on highway soil (HW 17 NB ramp, Deltaport Way, BC) using the soil amendments of lime and phosphate with three previously identified plant species, Lolium perenne L, Festuca rubra L and Poa pratensis L, both individually and in combination. This research addressed the phytostabilisation of metals along highway soils subjected to multi-metal additions by continuous highway runoff. The research tasks included: (1) quantifying the seasonal extent of metal accumulation in soil and assessing the seasonal impact on the metal speciation in the soil by the influence of soil amendments and different plant species; (2) determining accumulation differences between sampling periods in plant parts and to identify the plant part which accumulates significantly higher amounts of metals seasonally; and (3) assessing the influence of root-soil interactions on metal dynamics. The final outcome of the study helped in the development of a remediation strategy for metals (Cu, Pb, Mn and Zn) involving suitable plants and amendments, incorporating seasonal and rhizosphere influences and maintaining the functional and biological integrity of soil after remediation. A research paper based on this work is presented in Chapter 8.  1.4 Research contributions  This project is of significant practical and scientific relevance, since the results allow the effects of plant growth and amendment addition on phytostabilisation of metals in highway soils with different multi-metal contamination levels to be assessed. The results highlight the importance in identifying Best Management Practices (BMP) for phytostabilisation of metals (Cu, Pb, Mn and Zn) along highway soils. Plants suitable for phytoextraction and phytostabilisation of different metal contaminants were identified. The stage of plant growth suitable for maximum metal immobilisation, the seasonal impact on metal partitioning in soil and in plants and the effect of rhizosphere chemistry on metal dynamics were investigated, providing necessary information to formulate guidelines for metal remediation of moderately contaminated acid soils. By phytostabilisation, since the metal contaminants are mostly retained in the below ground portion of the vegetation, the transfer or transport of contaminants to the surrounding environment is lessened, reducing the chances of environmental pollution. Even if the plants die and roots disintegrate, the metal-contaminants still remain inactive in the soil as long as the soil physico chemical characteristics are not altered. Addition of lime as a stabilizing agent at the required  8 dose is environmentally friendly, since it has no harmful effects on either increasing the mobility of metals or the growth of associated ecological partners. Similarly the application of P can convert some of the metals, especially Pb to more immobile forms. The chances of eutrophication by P addition can occur only if P is present in a particular ionic form (mobile) which is unlikely for the soil pH created by lime application. Hence the suggested approach, a containment remediation strategy, not only alleviates existing risks, but also reduces the associated risks of metal effects. Inactivating metal contaminants at the source by using suitable plants and natural soil amendments helps to reduce exposure pathways for metal pollutants and contributes to ecological restoration. It also retains the functional and ecological integrity of soil after remediation. The results from these studies provided sufficient information to formulate specific guidelines for phytostabilisation of metal contaminants (Cu, Pb, Mn and Zn) in a highway soil with multi- metal contamination.  1.5 Dissertation organization  The dissertation is presented in nine chapters addressing specific aspects of metal remediation in soil-plant systems.  Chapter 1 provides a general introduction consisting of the problem statement, scope and objectives, research plan and research contributions of this study.  Chapter 2 is the review paper on "Phytoremediation Technology: Hyper-Accumulation Metals in Plants", published in Water, Air, and Soil Pollution.  Chapters 3 to 9 are journal papers, each of which is comprised of a brief introduction of the specific study, materials and methods adopted and presentation of the results, discussion and conclusions.  Chapter 3 focuses on preliminary examination of field measurements of metal accumulation (Cu, Pb, Mn and Zn) in soils and plants along highway sites and investigated the potential for phytostabilisation of plant species that spontaneously colonised the sites. A version of this  9 chapter was presented at the 9th International Conference on the Biogeochemistry of Trace Elements. (ICOBTE), Beijing, China, 2007 and published in “Biogeochemistry of Trace Elements: Environmental Protection, Remediation and Human Health”.  Chapters 4 and 5 focus on the first study, dealing with the identification of plant genera suitable for phytoextraction/phytostabilisation under British Columbia climatic conditions and soils. Chapter 4 describes the absorption characteristics and translocation properties of metals in different plant species at two growth stages, 90 and 120 DAS (days after sowing), published in “International Journal of Phytoremediation”. Chapter 5 deals specifically with the effect of plant growth on soil metal fractionation and total soil metal concentration at two plant growth stages, 90 and 120 DAS, published in “Land Reclamation and Contamination”.  Chapters 6 and 7 are focused on the second study, dealing with the effect of addition of soil amendments in modifying the soil properties and influencing the plants to immobilize metals. Chapter 6 describes the effect of plants and soil amendments on Cu and Zn immobilization, accepted for publication in “International Journal of Phytoremediation”. Chapter 7 describes the effect of plants and soil amendments on Pb and Mn immobilization, published in “Bioresource Technology”.  Chapter 8 reports the field study. It consists of the seasonal impact on metal accumulation in soil, soil metal fractionation at different seasons, seasonal influence on the metal accumulation and translocation in plants. It also discusses the influence of rhizosphere on soil metal dynamics.  Chapter 9 consists of general conclusions and recommendations of the various studies. It is followed by an appendix section (Appendix A to F), which provides additional information on study sites, experimental design, weather data during pot experiments and field study, QA/QC procedures and protocols, abstract of ANOVA for field experiment etc.  10 1.6 References  Adriano, D. C., Wenzel, W. W., Vangronsveld, J. and Bolan, N. S. (2004). Role of assisted natural remediation in environmental cleanup. Geoderma, 122, 121–142.  Alkorta, I., Herna´ndez-Allica, J., Becerril, J. M., Amezaga, I., Albizu, I. and Garbisu, C. (2004). Recent findings on the phytoremediation of soils contaminated with environmentally toxic heavy metals and metalloids such as zinc, cadmium, lead, and arsenic. Reviews in Environmental Science and Bio Technology, 3, 71–90.  Alloway, B. J. (1995). Soil processes and the behavior of metals. In: Alloway B. J. (Ed), Heavy metals in soils. London: Blackie, pp. 38–57.  Al-Chalabi, A. S. and Hawker, D. (2000). Distribution of vehicular lead in roadside soils of major roads of Brisbane, Australia. Water, Air, and Soil Pollution, 118, 299–310.  Asami, T. (1984). Pollution of soils by cadmium in changing metal cycles and human health. Ed. J O Nriagu Dahlem Konferezen, Berlin, Heidelberg, New York, Tokyo, Springer-Verlag.  Baker, A. J. M. and Brooks, R. R. (1989). Terrestrial higher plants which hyper accumulate metallic elements – Review of their distribution, ecology, and phytochemistry. Biorecovery, 1, 81–126.  Baudouin, C., Charveron, M., Tarrouse, R. and Gall, Y. (2002). Environmental pollutants and skin cancer. Cell Biology and Toxicology, 18, 341–348.  Birch, G. E. and Scollen, A. (2003). Heavy metals in road dust, gully pots and parkland soils in a highly urbanised subcatchment of Port Jackson, Australia. Australian Journal of Soil Research, 41, 1329–1342.  Chaney, R. L., Malik, M., Li, Y. M., Brown, S. L., Brewer, E. P. and Angle, J. S. (1997). Phytoremediation of soil metals. Current Opinion in Biotechnology, 8, 279–283.  Clemente, R., Almela, C. and Bernal, P. M. (2006). A remediation strategy based on active phytoremediation followed by natural attenuation in a soil contaminated by pyrite waste. Environmental Pollution, 143(3), 397-406.  Conesa, H. M., Faz, Á. and Arnaldos, R. (2006). Heavy metal accumulation and tolerance in plants from mine tailings of the semiarid Cartagena-La Unión mining district (SE Spain). Science of The Total Environment, 366, 1–11.  Environment Canada. (2003). Phytorem – Potential Green solutions for metal contaminated sites, green biotechnology, CD - rom.  Hamlin, R. L. and Parker, A. V. (2006). Phytoextraction potential of Indian mustard at various levels of zinc exposure. Journal of Plant Nutrition, 29, 1257–1272.   11 Hodes, G., Thomas, V. and Williams, A. (2003). A Strategy to Phase-Out Lead in African Gasoline. Renewable Energy for Development, Stockholm Environment Institute, 16(3).  Hughes, J. B., Shanks, J., Vanderford, M., Lauritzen, J. and Bhadra, R. (1997) Transformation of TNT by aquatic plants and plant tissue cultures. Environmental Science and Technology, 31, 266–271.  Kabata-Pendias, A. and Adriano, D. C. (1995). Trace metals in soil amendments and environmental quality, eds. J. E Rechcigl Lewis Publishers, New York, pp. 139-167.  Kumpiene, J., Lagerkvist, A. and Maurice, C. (2007). Stabilization of Pb- and Cu-contaminated soil using coal fly ash and peat. Environmental Pollution, 145, 365-373.  McGrath, S. P. (1987). Long-term studies of metal transfers following applications of sewage sludge. In Pollutant Transport and Fate in Ecosystems. Eds. P. J Coughtrey, M. H Martin and M. H Unsworth. Special Publication No. 6 of the British Ecological Society, Blackwell Scientific, Oxford, pp. 301–317.  Mench, M., Vangronsveld, J., Clijsters, H., Lepp, N. W. and Edwards, R. (2000): In situ metal immobilization and phytostabilization of contaminated soils. In: Terry N., Bañuelos G. (eds.): Phytoremediation of Contaminated Soil and Water. Lewis Publ., Boca Raton, London, New York, Washington D.C, pp. 323–358.  Meyers, D. E. R., Auchterlonie, G. J., Webb, R. I. and Wood, B. (2008). Uptake and localisation of lead in the root system of Brassica juncea. Environ. Pollut., 153 (2), 323-332.  Sansalone, J. J. and Buchberger, S. G. (1997). Partitioning and first flush of metals in urban roadway storm water. J. Environ. Eng., 123, 134–143.  Sezgin, N., Ozcan, H. K., Demir, G., Nemlioglu, S. and Bayat, C. (2003). Determination of heavy metal concentrations in street dusts in Istanbul E-5 highway. Environment International, 29, 979–985.  Simon, L. (2005) Stabilization of metals in acidic mine spoil with amendments and red fescue (Festuca rubra L.) growth. Environ. Geochem. Health, 27, 289–300.  Smith, R. A. H. and Bradshaw, A. D. (1979). The use of metal tolerant plant populations for the reclamation of metalliferous wastes. Journal of Applied Ecology, 16, 595–612.  Smolders, E. and Degryse, F. (2002). Fate and effect of zinc from tire debris in soil. Environmental Science and Technology, 36, 3706- 3710.  Sutherland, R. A., Day, J. P. and Bussen, J. O. (2003). Lead concentrations, isotope ratios and source apportionment in road deposited sediments, Honolulu, Oahu, Hawaii. Water, Air, and Soil Pollution, 142, 165-186.  Turner, R. E., Swenson, E. M. and. Milan, C. S. (2001). Organic and inorganic contributions to vertical accretion in salt marsh sediments, In M. Weinstein and K. Kreeger [eds.], Concepts and controversies in tidal marsh ecology. Kluwer, pp. 583–595.  12  Varrica, D., Dongarra, G., Sabatino, G. and Monna, F. (2003). Inorganic geochemistry of roadway dust from the metropolitan area of Palermo, Italy. Environmental Geology, 44, 222-230.  Viklander, M. (1998). Particle size distribution and metal content in street sediments. Journal of Environmental Engineering, 124, 761-766.  Wang, A. S., Angle, J. S., Chaney, R. L., Delorme, T. A. and Reeves, R. D. (2006). Soil pH effects on uptake of Cd and Zn by Thlaspi caerulescens. Plant and Soil, 281(1–2), 325– 337.  Weng, G., Wu, L., Wang, Z., Luo, Y. and Christie, P. (2005). Copper uptake by four Elsholtzia ecotypes supplied with varying levels of copper in solution culture. Environment International, 31 (6), 880-884.  Zayed, J., Guessous, A., Lambert, J., Carrier, G. and Philippe, S. (2003). Estimati.on of annual Mn emissions from MMT source in the Canadian environment and the Mn pollution index in each province. Science of The Total Environment, 312(1-3), 147-154.  Zhu, Y., Yu, H., Wang, J., Fang, W., Yuan, J. and Yang, Z. (2007). Heavy metal accumulations of 24 asparagus bean cultivars grown in soil contaminated with Cd alone and with multiple metals (Cd, Pb, and Zn). J. Agric. Food Chem., 55, 1045-1052. `  13 2. 1PHYTOREMEDIATION TECHNOLOGY: HYPER-ACCUMULATION METALS IN PLANTS   2.1 Introduction  Heavy metals are ubiquitous environmental contaminants in industrialized societies. Soil pollution by metals differs from air or water pollution, because metals persist in soil much longer than in other compartments of the biosphere (Lasat, 2002). Over recent decades, the annual worldwide release of metals reached 22,000 t (metric ton) for cadmium, 939,000 t for copper, 783,000 t for lead and 1,350,000 t for zinc (Singh et al., 2003). Sources of metal contaminants in soils include metalliferous mining and smelting, metallurgical industries, sewage sludge treatment, warfare and military training, waste disposal sites, agricultural fertilizers and electronic industries (Alloway, 1995). For example, mine tailings rich in sulphide minerals may form acid mine drainage (AMD) through reaction with atmospheric oxygen and water, and AMD contains elevated levels of metals that could be harmful to animals and plants (Stoltz, 2004).  Ground-transportation also causes metal contamination. Highway traffic, maintenance, and de-icing operations generate continuous surface and groundwater contaminant sources. Tread ware, brake abrasion, and corrosion are well documented heavy metal sources associated with highway traffic (Ho and Tai, 1988; Fatoki, 1996; Garcia and Millan, 1998; Sanchez- Martin et al., 2000). Heavy metal contaminants in roadside soils originate from engine and brake pad wear (e.g. Cd, Cu, and Ni) (Viklander, 1998); lubricants (e.g. Cd, Cu and Zn) (Birch and Scollen, 2003, Turer et al., 2001); exhaust emissions, (e.g. Pb) (Gulson et al., 1981; Al-Chalabi and Hawker, 2000; Sutherland et al., 2003); and tire abrasion (e.g. Zn) (Smolders and Degryse, 2002). The concentration ranges of metals of greatest importance in roadside soils are given in Figure 2.1.     1  A version of this chapter has been published. Padmavathiamma, P.K. and Li, L.Y. (2007) Phytoremediation Technology: Hyper-accumulation metals in plants. Water Air Soil Pollution, 184: 105-126.   14                             Figure Metal Regression Equation, y Correlation, R (a) Mn 64.487x0.1919 0. 495  Zn 25.616x-0.427 0.449  Pb 31.996x-0.0989 0.189 (b) Pb 347.5x-0.8549 0.983  Cu 43.347x-0.3368 0.981  Zn 110.66x-0.3295 0.998 (c) Pb 319.69x-1.1831 0.909  Cu 197.25x-1.0689 0.925  Zn 271.6x-0.6321 0.897 (d) Pb 206.93x-0.6 0.986  Zn 227.69x-0.1842 0.871  Figure 2.1  Heavy metal content of road-side soils from (a) Brussels-Ortend, Belgium (Albasel and Cottenie, 1985); (b) Osogobo, Nigeria (Fakayode and Olu-Owolabi, 2003); (c) West bank, Palestine (Swaileh et al., 2004); (d) A31 between Nancy and France (Viard et al., 2004).  0 20 40 60 80 100 120 140 160 0 5 10 15 Distance from the Highway (m) Co n ce n tr at io n  (m g/ kg )    . Mn Zn Pb Power (Mn) Power (Pb) Power (Zn) (a) 0 25 50 75 100 0 10 20 30 40 50 60 Distance from the Highway (m) Co n ce n tr at io n  (m g/ kg )    . Pb Cu Zn Pow er (Pb) Pow er (Zn) Pow er (Cu) (b) 0 50 100 150 200 250 300 350 0 5 10 15 20 25 30 35 Distance from the Highway (m) Co n ce n tr at io n  (m g/ kg )    . . Pb Cu Zn Pow er (Pb) Pow er (Cu) Pow er (Zn) (c) 0 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350 Distance from the highway (m) Co n ce n tr at io n  (m g/ kg )    . . Pb Zn Pow er (Pb) Pow er (Zn) (d)  15  Toxic heavy metals cause DNA damage, and their carcinogenic effects in animals and humans are probably caused by their mutagenic ability (Knasmuller et al., 1998; Baudouin et al., 2002). Exposure to high levels of these metals has been linked to adverse effects on human health and wildlife. Lead poisoning in children causes neurological damage leading to reduced intelligence, loss of short term memory, learning disabilities and coordination problems. The effects of arsenic include cardiovascular problems, skin cancer and other skin effects, peripheral neuropathy (WHO, 1997) and kidney damage. Cadmium accumulates in the kidneys and is implicated in a range of kidney diseases (WHO, 1997). The principal health risks associated with mercury are damage to the nervous system, with such symptoms as uncontrollable shaking, muscle wasting, partial blindness, and deformities in children exposed in the womb (WHO, 1997).  Metal-contaminated soil can be remediated by chemical, physical or biological techniques (McEldowney et al., 1993). Chemical and physical treatments irreversibly affect soil properties, destroy biodiversity and may render the soil useless as a medium for plant growth. These remediation methods can be costly. Table 2.1 summarizes the cost of different remediation technologies.  Table 2.1. Cost of different remediation technologies (Glass, 1999).  Process Cost($/ton) Other factors Vitrification 75-425 Long-term monitoring Land filling 100-500 Transport/excavation/monitoring Chemical treatment 100-500 Recycling of contaminants Electrokinetics 20-200 Monitoring Phytoextraction 5-40 Disposal of phytomass  Among the listed remediation technologies, phytoremediation is one of the lowest cost techniques for contaminated soil remediation. There is a need to develop suitable cost-effective biological soil remediation techniques to remove contaminants without affecting soil fertility. Phytoremediation could provide sustainable techniques for metal remediation. This paper  16 summarizes the development of phytoremediation for metals in the past two decades. Phytoremediation involves the use of plants to remove, transfer, stabilize and/or degrade contaminants in soil, sediment and water (Hughes et al., 1997). The idea that plants can be used for environmental remediation is very old and cannot be traced to any particular source. The concentration of metal uptake in plants is shown in Figure 2.2. A series of fascinating scientific discoveries, combined with interdisciplinary research, has allowed phytoremediation to develop into a promising, cost-effective, and environmentally friendly technology.  The term phytoremediation ("phyton" meaning plant, and the Latin suffix "remedium" meaning to clean or restore) refers to a diverse collection of plant-based technologies that use either naturally occurring, or genetically engineered, plants to clean contaminated environments (Cunningham et al., 1997; Flathman and Lanza, 1998). Some plants which grow on metalliferous soils have developed the ability to accumulate massive amounts of indigenous metals in their tissues without symptoms of toxicity (Reeves and Brooks, 1983; Baker and Brooks, 1989; Baker et al., 1991; Entry et al., 1999). The idea of using plants to extract metals from contaminated soil was re-introduced and developed by Utsunamyia (1980) and Chaney (1983). The first field trial on Zn and Cd phytoextraction was conducted by Baker et al. (1991).  Several comprehensive reviews have been written, summarizing many important aspects of this novel plant-based technology (Salt et al., 1995, 1998; Chaney et al., 1997; Raskin et al., 1997; Chaudhry et al., 1998; Wenzel et al., 1999; Meagher, 2000; Navari-Izzo and Quartacci, 2001; Lasat, 2002; McGrath et al., 2002; McGrath and Zhao, 2003; McIntyre, 2003; Singh et al., 2003; Garbisu and Alkortha, 2001; Prasad and Freitas, 2003; Alkortha et al., 2004; Ghosh and Singh, 2005; Pilon-Smits, 2005). These reviews give general guidance and recommendations for applying phytoremediation, highlighting the processes associated with applications and underlying biological mechanisms. The present review is intended to give an updated, more concise version of information so far available with respect to different subsets of phyoremediation. It provides a critical overview of the present state of the art, with particular emphasis on phytoextraction and phytostabilization of soil heavy metal contaminants.       17                                       Figure 2.2  Heavy metal content in plants growing on contaminated sites (Yoon et al., 2006). (a) Bahia grass (Paspalum notatum); (b) Wire grass (Gentiana pennelliana); (c) Ticktrefoil (Desmodium paniculatum); (d) Flats edge (Cyperus esculentus); (e) Bermuda grass (Cynodon dactylon).  .    0 50 100 150 200 250 Pb Cu Zn Heavy metals Co n ce n tr at io n (m g/ kg ) (c) 0 50 100 150 200 250 300 350 Pb Cu Zn Heavy metals Co n ce n tr at io n (m g/ kg ) (d) 0 200 400 600 800 1000 1200 1400 1600 1800 2000 Pb Cu Zn Heavy metals Co n ce n tr at io n (m g/ kg ) ShootRoot Soil (e) 0 200 400 600 800 1000 1200 Pb Cu Zn Heavy metals Co n ce n tr at io n (m g/ kg ) (b) 0 200 400 600 800 1000 1200 1400 1600 Pb Cu Zn Heavy metals Co n ce n tr at io n (m g/ kg ) (a)  18 2.2. Categories of phytoremediation  Depending on the contaminants, the site conditions, the level of clean-up required, and the types of plants, phytoremediation technology can be used for containment (phytoimmobilization and phytostabilization) or removal (phytoextraction and phytovolatilization) purposes (Thangavel and Subhuram, 2004). The four different plant-based technologies of phytoremediation, each having a different mechanism of action for remediating metal-polluted soil, sediment, or water: (1) phytostabilization, where plants stabilize, rather than remove contaminants by plant root metal retention; (2) phytofiltration, involving plants to clean various aquatic environments; (3) phytovolatilization, utilizing plants to extract certain metals from soil and then release them into the atmosphere by volatilization; and (4) phytoextraction, in which plants absorb metals from soil and translocate them to harvestable shoots where they accumulate. The different mechanisms of phytoremediation are summarized in Table 2.2.  Ecological issues also need to be evaluated when developing a phytoremediation strategy for a polluted site. In particular, one has to consider how the phytoremediation efforts might affect local ecological relationships, especially those involving other crops. Since the phytoremediation plants will be grown under contaminated soil/ water conditions, where other crops may not thrive because of contaminant toxicities, the competition problem is unlikely to arise.  Table 2.2 Different mechanisms of phytoremediation (Ghosh and Singh, 2005)  Process Mechanisms Contaminant Phytofiltration Rhizosphere accumulation Organics/ Inorganic Phytostabilisation Complexation Inorganic Phytoextraction Hyper accumulation Inorganic Phytovolatilization Volatilisation by leaves Organics/ Inorganic     19 2.2.1 Phytostabilization  Phytostabilization uses certain plant species to immobilize contaminants in soil, through absorption and accumulation by roots, adsorption onto roots or precipitation within the root zone and physical stabilization of soils. The schematic mechanism of phytostabilization is illustrated in Figure 2.3.  This process reduces the mobility of contaminants and prevents migration to groundwater or air. This can re-establish a vegetative cover at sites where natural vegetation is lacking due to high metal concentrations (Tordoff et al., 2000). Thorough planning is essential for successful revegetation, including physical and chemical analyses, bioassays and field trials. The main approaches to revegetation are summarized in Table 2.3. Metal-tolerant species may be used to restore vegetation to such sites, thereby decreasing the potential migration of contaminants through wind, transport of exposed surface soils, leaching of soil and contamination of groundwater (Stoltz and Greger, 2002).     Figure 2.3 Schematic mechanism of phytostabilization.  Unlike other phytoremediative techniques, phytostabilization is not intended to remove metal contaminants from a site, but rather to stabilize them by accumulation in roots or precipitation within root zones, reducing the risk to human health and the environment. It is applied in situations where there are potential human health impacts, and exposure to substances of concern can be reduced to acceptable levels by containment. The disruption to site activities may be less than with more intrusive soil remediation technologies.  20 Phytostabilization is most effective for fine-textured soils with high organic matter content, but it is suitable for treating a wide range of sites where large areas are subject to surface contamination (Cunningham et al., 1997; Berti and Cunningham, 2000). However, some highly contaminated sites are not suitable for phytostabilization, because plant growth and survival is impossible (Berti and Cunningham, 2000). Phytostabilization has advantages over other soil-remediation practices in that it is less expensive, easier to implement, and preferable aesthetically. (Berti and Cunningham, 2000; Schnoor, 2000). When decontamination strategies are impractical because of the extent of the contaminated area or the lack of adequate funding, phytostabilization is advantageous (Berti and Cunningham, 2000). It may also serve as an interim strategy to reduce risk at sites where complications delay the selection of the most appropriate technique.  Table 2.3. Approaches to revegetation (adapted from Williamson and Johnson, 1981)  Soil characteristics Reclamation technique Problems encountered Low toxicity- total metal content <0.1%. Amelioration and direct seeding with grasses and legumes. Seed or transplant ecologically adapted native species. Apply lime, organic matter and fertilizers as necessary Medium or long –term maintenance program. Expertise required on the characteristics of native flora. Grazing must be strictly monitored and excluded in some situations High toxicity-total metal content > 0.1%. Amelioration and direct seeding with metal tolerant and salt tolerant (saline) ecotypes. Apply lime, organic matter and fertilizers as necessary. Amelioration with 10-50 cm of innocuous mineral waste and organic material and seeding with grasses and legumes. Apply lime and fertilizer if necessary Commitment to regular management. Expertise required for the selection of tolerant ecotypes. Grazing management not possible. Regression will occur if depths of amendment are shallow or if upward movement of metals occurs. Availability and transport costs limiting. Extreme toxicity Isolation; surface treatment with 30-100 cm of innocuous barrier material and surface banding with10-30 cm of rooting medium. Apply lime and fertilizer if necessary. High cost and potential limitation of material availability.      21  Characteristics of plants appropriate for phytostabilization at a particular site include: tolerance to high levels of the contaminant(s) of concern; high production of root biomass able to immobilize these contaminants through uptake, precipitation, or reduction; and retention of applicable contaminants in roots, as opposed to transfer to shoots, to avoid special handling and disposal of shoots.  Yoon et al. (2006) evaluated the potential of 36 plants (17 species) growing on a contaminated site and found that plants with a high bio-concentration factor (BCF, metal concentration ratio of plant roots to soil) and low translocation factor (TF, metal concentration ratio of plant shoots to roots) have the potential for phytostabilization (Figure 2.2, a-e). The lack of appreciable metals in shoot tissue also eliminates the necessity to treat harvested shoot residue as a hazardous waste (Flathman and Lanza, 1998). In a field study, mine wastes containing copper, lead, and zinc were stabilized by grasses (Agrostis tenuis cv. Goginan for acid lead and zinc mine wastes, Agrostis tenuis cv. Parys for copper mine wastes, and Festuca rubra cv. Merlin for calcareous lead and zinc mine wastes) (Smith and Bradshaw, 1992). The research of Smith and Bradshaw (1992) led to the development of two cultivars of Agrostis tenuis Sibth and one of Festuca rubra L which are now commercially available for phytostabilizing Pb-, Zn-, and Cu-contaminated soils.  Stabilization also involves soil amendments to promote the formation of insoluble metal complexes that reduce biological availability and plant uptake, thus preventing metals from entering the food chain (Adriano et al., 2004; Berti and Cunningham, 2000; Cunningham et al., 1997). One way to facilitate such immobilisation is by altering the physicochemical properties of the metal-soil complex by introducing a multipurpose anion, such as phosphate, that enhances metal adsorption via anion-induced negative charge and metal precipitation (Bolan et al., 2003). Addition of humified organic matter (O.M.) such as compost, together with lime to raise soil pH (Kuo et al., 1985), is a common practice for immobilizing heavy metals and improving soil conditions, to facilitate re-vegetation of contaminated soils (Williamson and Johnson, 1981). Soil acidification, due to the oxidation of metallic sulphides in the soil, increases metal bioavailability; but liming can control soil acidification; and organic materials generally promote fixation of heavy metals in non-available soil fractions, with Cu bioavailability being particularly affected by organic treatments (Clemente et al., 2003). The production of sulphate  22 by sulphide oxidation increases solubility of Zn and Mn, and therefore their concentrations in plant-available (DTPA-extractable) fractions. However, the bioavailability of Cu did not decrease with either soil pH increase or with lime, indicating that the organic treatments might have had a significant effect. Revegetation of mine tailings usually requires amendments of phosphorus, even though phosphate addition can mobilize arsenic (As) from the tailings. Recent research results on phytostabilization are summarized in Table 2.4.  Table 2.4. Summary of research results - Phytostabilisation  Plant species Metal Treatments Results Limitations Reference Hordeum vulgare, Lupinus angustifolius, Secale cereale As Different P amendment products (organic and inorganic) P amendment of < 3 g m-2 caused As leaching of 0.5 mg L-1 from unplanted lysimeters and up to 0.9 mg L-1 on average in planted lysimeters. Arsenic accumulated in plant biomass to 126 mg/kg in shoots and 469 mg/kg in roots. Variable species- amendment combinations produced differences in the amount of As leached and uptake. Mains et al., 2006a,b Lolium italicum and Festuca arundinaceae Pb and Zn Compost at two rates (10%, and 30% v/v) The concentration of Pb and Zn in aerial parts and in roots of L. italicum and F. arundinacea decreased more than five times in presence of compost. Pb content decreased from 218 to 32 mg/kg in shoot and 7232 to 1196 mg/kg in root. Zn decreased from 4190 to 624 mg/kg in shoot and 7120 to 1993 mg/kg in root. The level of contaminants in aerial parts of plants was still too high to be grazed by herbivores. Rizzi et al., 2004 Brassica juncea Cd Soil amendments- liming materials, phosphate compounds and biosolids Phosphate immobilized Cd, thereby reducing the phytotoxicity of Cd. The tissue metal concentration of Cd, Cu and Cr(VI) with biosolids application was 253, 157 and 12.4 mg/kg. (i.e. a decrease over nil amendment).  Bolan et al., 2003        23 Plant species Metal Treatments Results Limitations Reference Brassica juncea Zn, Cu, Mn, Fe, Pb and Cd  organic amendments (cow manure and compost) and lime Active phytoremediation followed by natural attenuation, was effective for remediation of pyrite- polluted soil. Bioavaialble soil concentration decreased from: 363 to 166 mg/kg for Zn, 36 to 31 mg/kg for Cu, 1.94 to 1.48 mg/kg for Pb, 1.6 to 0.86 mg/kg for Cd, 679 to 303 mg/kg for Fe and 245 to 120 mg/kg for Mn. Available As concentration in soil decreased from 2.5-13.5 mg/kg after the first crop to 0.5-2.6 mg/kg after the second. Bioavailability of Cu did not decrease with either soil pH increase or with lime. Clemente et al., 2003, Clemente et al., 2006 Hyparrhenia hirta and Zygophyllum fabago Pb, Zn and Cu Characterizat ion of soil and plant samples from a mine tailing located in South-East Spain for further phytostabilisa tion research H. hirta accumulated around 150 mg kg−1 Pb in both shoots and roots. Zn concentration was 750 mg kg−1 in Z. fabago shoots. The plant species, H. hirta and Z. fabago, colonize only parts of the tailings with low electrical conductivity Conesa et al., 2006  Leachates and uptakes of As were found to be higher with an organic fertilizer amendment than super-phosphate, particularly in combination with barley (Mains et al., 2006b). Active phytoremediation followed by natural attenuation, was effective for remediation of the pyrite- polluted soil (Clemente et al., 2006).  The Met PAD IM bio test was used to assess the extent of metal accumulation by plants in mining areas. Plants were identified as hyper tolerant which can be used for phytostabilization (Boularbah et al., 2006). Two plant species, Hyparrhenia hirta and Zygophyllum fabago, that have naturally colonized some parts of mine tailings in South-East Spain, have been reported to tolerate high metal concentrations in their rhizospheres. These plant species do not take up high  24 concentrations of metals, providing a good tool to achieve surface stabilization of tailings with low risk of affecting the food chain (Conesa et al., 2006).  Phytostabilization efforts in the Mediterranean region have been found to be improved by using mixtures including local metallicolous legume and grass species (Fre´rot et al., 2006). It is better to identify the plants spontaneously colonizing the contaminated site, since they are more ecologically adapted than introduced species.  2.2.2 Phytofiltration  Phytofiltration is the use of plant roots (rhizofiltration) or seedlings (blastofiltration) to absorb or adsorb pollutants, mainly metals, from water and aqueous-waste streams (Prasad and Freitas, 2003). Plant roots or seedlings grown in aerated water absorb, precipitate and concentrate toxic metals from polluted effluents (Dushenkov and Kapulnik, 2000; Elless et al., 2005). Mechanisms involved in biosorption include chemisorption, complexation, ion exchange, micro precipitation, hydroxide condensation onto the biosurface, and surface adsorption (Gardea-Torresdey et al., 2004).  Rhizofiltration uses terrestrial plants instead of aquatic plants because the former feature much larger fibrous root systems covered with root hairs with extremely large surface areas. Metal pollutants in industrial-process water and in groundwater are most commonly removed by precipitation or flocculation, followed by sedimentation and disposal of the resulting sludge (Ensley, 2000). The process involves raising plants hydroponically and transplanting them into metal-polluted waters where plants absorb and concentrate the metals in their roots and shoots (Dushenkov et al., 1995; Salt et al., 1995; Flathman and Lanza, 1998; Zhu et al., 1999). Root exudates and changes in rhizosphere pH may also cause metals to precipitate onto root surfaces. As they become saturated with the metal contaminants, roots or whole plants are harvested for disposal (Flathman and Lanza, 1998; Zhu et al., 1999).  Dushenkov et al. (1995), Salt et al. (1995), and Flathman and Lanza (1998) contend that plants for phytofiltration should accumulate metals only in the roots. Dushenkov et al. (1995) explain that the translocation of metals to shoots would decrease the efficiency of rhizofiltration by increasing the  25 amount of contaminated plant residue needing disposal. However, Zhu et al. (1999) suggest that the efficiency of the process can be increased by using plants with a heightened ability to absorb and translocate metals.  Several aquatic species have the ability to remove heavy metals from water, including water hyacinth (Eichhornia crassipes., Kay et al., 1984; Zhu et al., 1999), pennywort (Hydrocotyle umbellata L., Dierberg et al., 1987), and duckweed (Lemna minor L., Mo et al., 1989). However, these plants have limited potential for rhizofiltration because they are not efficient in removing metals as a result of their small, slow-growing roots (Dushenkov et al., 1995). The high water content of aquatic plants complicates their drying, composting, or incineration. In spite of limitations, Zhu et al. (1999) indicated that water hyacinth is effective in removing trace elements in waste streams. Sunflower (Helianthus annus L.) and Indian mustard (Brassica juncea Czern.) are the most promising terrestrial candidates for removing metals from water. The roots of Indian mustard are effective in capturing Cd, Cr, Cu, Ni, Pb, and Zn (Dushenkov et al., 1995), whereas sunflower removes Pb (Dushenkov et al., 1995), U (Dushenkov et al., 1997a), 137Cs, and 90Sr (Dushenkov et al., 1997b) from hydroponic solutions. A novel phytofiltration technology has been proposed by Sekhar et al. (2004) for removal and recovery of lead (Pb) from wastewaters. This technology uses plant-based biomaterial from the bark of the plant commonly called Indian sarsaparilla (Hemidesmus indicus). The target of their research was polluted surface water and groundwater at industrially contaminated sites. Cassava waste biomass was also effective in removing two divalent metal ions, Cd (II) and Zn (II), from aqueous solutions (Horsfall and Abia, 2003). Modification of the cassava waste biomass by treating it with thioglycollic acid resulted in increased adsorption rates for Cd, Cu, and Zn (Abia et al., 2003). Several species of Sargassum biomass (non living brown algae) were effective biosorbents for heavy metals such as Cd and Cu (Davis, et al., 2000).        26 Table 2.5. Summary of research results - Phytofiltration  Plant species Metal Treatments Results Reference Brassica juncea, Helianthus annuus Cu, Cd, Cr, Ni, Pb, and Zn  Roots of hydroponically grown terrestrial plants used to remove toxic elements from aqueous solutions Roots of B. juncea concentrated these metals 131-563-fold (on a DW basis) above initial solution concentrations. The recoveries of heavy metals were 45 % for Cd, 55% for Zn, 50% for Cr, 45% for Ni, 97% for Cu and 100 % for Pb. Dushenkov et al., 1995 Sunflower  U Rhizofiltration of U in water by roots of sunflower plants U concentration in water reduced from 21-874 ug/l to <20 ug/l by rhizofiltration Dushenkov et al., 1997 Water Hyacinth As, Cd Cr, Cu, Ni, and Se The abilities of water hyacinth to take up and translocate six trace elements-As, Cd, Cr, Cu, Ni, and Se were studied under controlled conditions The highest levels of Cd in shoots and roots were 371 and 6103 mg/kg dry wt., and those of Cr were 119 and 3951 mg/kg dry wt., Cadmium, Cr, Cu, Ni, and As were more highly accumulated in roots, whereas Se accumulated more in shoots. Zhu et al., 1999 Duckweed Hg Effects of pH, copper and humic acid Duckweed strongly absorbed Hg from water and after 3 days contained 2000 ppm of Hg by weight Mo et al., 1989 Duckweed (Lemna minor L.) and water velvet (Azolla pinnata). Fe and Cu Solutions enriched with 1·0, 2·0, 4·0, and 8·0 ppm of these 2 metal ions, renewed every 2 days over a 14-day test period. When duckweed was kept in a solution containing Cu alone at 8·0 ppm level, the value of the metal concentration factor after 14 days was 51.20. However, in the presence of an equal concentration of Fe the value of this factor was 26.53, indicating the influence of Fe on the uptake rate of Cu. Jain et al., 1989   27 Plants used for phytofiltration should be able to accumulate and tolerate significant amounts of the target metals, in conjunction with easy handling, low maintenance costs, and a minimum of secondary waste requiring disposal. It is also desirable for plants to produce significant amounts of root biomass or root surface area (Dushenkov and Kapulnik, 2000). Reports on phytofiltration are summarized in Table 2.5.  2.2.3 Phytovolatilization  Some metal contaminants such as As, Hg, and Se may exist as gaseous species in the environment. In recent years, researchers have sought naturally-occurring or genetically- modified plants capable of absorbing elemental forms of these metals from the soil, biologically converting them to gaseous species within the plant, and releasing them into the atmosphere. This process is called phytovolatilization. The mechanism of phytovolatilization is shown schematically in Figure 2.4.   Figure 2.4 Schematic mechanism of phytovolatilization.  Volatilization of Se from plant tissues may provide a mechanism of selenium detoxification. As early as 1894, Hofmeister proposed that selenium in animals is detoxified by releasing volatile dimethyl selenide from the lungs, based on the fact that the odour of dimethyl telluride was detected in the breath of dogs injected with sodium tellurite. Using the same logic, it was suggested that the garlicky odour of plants that accumulate selenium may indicate release of  28 volatile selenium compounds. This is the most controversial of phytoremediation technologies. Hg and Se are toxic (Suszcynsky and Shann, 1995), and there is doubt about whether the volatilization of these elements into the atmosphere is desirable or safe (Watanabe, 1997).  The volatile selenium compound released from the selenium accumulator Astragalus racemosus was identified as dimethyl diselenide (Evans et al., 1968). Selenium released from alfalfa, a selenium nonaccumulator, was different from the accumulator species and was identified as dimethyl selenide. Lewis et al. (1966) showed that both selenium nonaccumulator and accumulator species volatilize selenium. Selenium phytovolatilization has received the most attention to date (Lewis et al., 1966; Terry et al., 1992; Bañuelos et al., 1993; McGrath, 1998) because this element is a serious problem in many parts of the world where there are Se-rich soils (Brooks, 1998). According to Brooks (1998), the release of volatile Se compounds from higher plants was first reported by Lewis et al. (1966). Terry et al. (1992) report that members of the Brassicaceae are capable of releasing up to 40 g Se ha-1 day -1 as various gaseous compounds. Some aquatic plants, such as cattail (Typha latifolia L.), have potential for Se phytoremediation (Pilon-Smits et al., 1999).  Volatile Se compounds such as dimethyl selenide are 1/600 to 1/500 as toxic as inorganic forms of Se found in soil (DeSouza et al., 2000). The volatilization of Se and Hg is also a permanent site solution, because the inorganic forms of these elements are removed, and gaseous species are not likely to redeposit at or near the site (Atkinson et al., 1990; Heaton et al., 1998). Furthermore, sites that utilize this technique may not require much management after the original planting. This remediation method has the added benefits of minimal site disturbance, less erosion, and no need to dispose of contaminated plant material (Heaton et al., 1998). Heaton et al. (1998) suggest that the transfer of Hg (O) to the atmosphere would not contribute significantly to the atmospheric pool. This technique appears to be a promising tool for remediating Se- and Hg- contaminated soils.  Volatilization of arsenic as dimethyl arsenite has also been postulated as a resistance mechanism in marine algae. However, it is not known whether terrestrial plants also volatilize arsenic in significant quantities. Studies on arsenic uptake and distribution in higher plants indicate that arsenic predominantly accumulates in roots and that only small quantities are transported to  29 shoots. However, plants may enhance the biotransformation of arsenic by rhizospheric bacteria, thus increasing the rates of volatilization (Salt et al., 1998).  Unlike other remediation techniques, once contaminants have been removed via volatilization, there is a loss of control over their migration to other areas. Some authors suggest that the addition to atmospheric levels through phytovolatilization would not contribute significantly to the atmospheric pool, since the contaminants are likely to be subject to more effective or rapid natural degradation processes such as photodegradation (Azaizeh et al., 1997). However, phytovolatilization should be avoided for sites near population centres and at places with unique meteorological conditions that promote the rapid deposition of volatile compounds (Heaton et al., 1998).  Hence the consequences of releasing the metals to the atmosphere need to be considered carefully before adopting this method as a remediation tool.  2.2.4 Phytoextraction  Phytoextraction, also called phytoaccumulation, refers to the uptake and translocation of metal contaminants in the soil by plant roots into above-ground components of the plants (Figure 2.5). The typical levels of metal concentration effects in plants are given in Table 2.6.   Figure 2.5 Schematic mechanism of phytoextraction.  30   Table 2.6 Effect of typical levels for metals in plants   Status Metal concentrations (mg/kg)  Cd Cu Pb Zn Deficient – <1–5 – <10 Normal 0.05–2 3–30 0.5–10 10–150 Phytotoxic 5–700 20–100 30–300 >100  Adapted from Pugh et al. (2002)   Table 2.7(a). Examples of hyperaccumulators and their bioaccumulation potential.  Plant species Metal Content (mg/kg) Reference Thlaspi caerulescens Zn 39,600 (shoots) Reeves and Brooks (1983) Thlaspi caerulescens Cd 1800 Baker and Walker (1990) Ipomea alpine Cu 12300 Baker and Walker (1990) Sebertia acuminate Ni 25% by wt. dried sap Jaffre et al. (1976) Haumaniastrum robertii Co 10,200 Brooks (1998) Astragalus racemosus Se 14 900 Beath et al. (1937) Pteris vittata As 27,000 Wang et al, 2002 Berkheya coddii Ni 5500 Robinson et al., 1997 Iberis intermedia Ti 3070 Leblanc et al., 1999       31  Table 2.7 (b) Hyperaccumulators and their bioaccumulation potential.  Plant species Metal Results Reference Pistia stratiotes Ag, Cd, Cr, Cu, Hg, Ni, Pb and Zn. All elements accumulated mainly in the root system. Odjegba and Fasidi, 2004 Spartina plants Hg Organic Hg was absorbed and transformed into an inorganic form (Hg+, Hg2+) and accumulated in roots. Tian et al., 2004 Helianthus annuus Pb Pb concentrated in the leaf and stem indicating the prerequisites of a hyperaccumulator plant. Boonyapookana et al., 2005 Hemidesmus indicus Pb Heavy metal mainly accumulated in roots and shoots. Chandra et al., 2005 Sesbania drummondii Pb Pb accumulated as lead acetate in roots and leaves, although lead sulfate and sulfide were also detected in leaves, whereas lead sulfide was detected in root samples. Lead nitrate in the nutrient solution biotransformed to lead acetate and sulfate in its tissues. Complexation with acetate and sulfate may be a lead detoxification strategy in this plant species. Sharma et al., 2004 Lemna gibba As A preliminary bioindicator for As transfer from substrate to plants. Used for As phytoremediation of mine tailing waters because of its high accumulation capacity. Mkandawire and Dudel, 2005 Pteris vittata, P. cretica, P. longifolia and P. umbrosa. As Suitable for phytoremediation in the moderately contaminated soils. Caille et al., 2004  Alyssum Ni Majority of Ni is stored either in the leaf epidermal cell vacuoles, or in the basal portions of the numerous stellate trichomes. The metal concentration in the trichome basal compartment was the highest ever reported for healthy vascular plant tissue, approximately 15-20% dry weight. Broadhurst et al., 2004 Solanum nigrum and Conyza canadensis Cd High concentration of Cd accumulated. Tolerant to combined action of Cd, Pb, Cu and Zn Wei et al., 2004  32 Plant species Metal Results Reference Thlaspi caerulescens Cd High Cd-accumulating capability, acquiring Cd from the same soil pools as non-accumulating species. Schwartz et al., 2003 Arabis gemmifera Cd and Zn Hyperaccumulator of Cd and Zn, with phytoextraction capacities almost equal to Thlaspi caerulescens. Kubota and Takenaka, 2003 Stanleya pinnata Se Adapted to semi-arid western U. S. soils and environments. Uptake, metabolism and volatilization of Se. Parker et al., 2003 Austromyrtus bidwilli  . Phytolacca acinosa Roxb Mn Australian native hyperaccumulator of Mn, grows rapidly, has substantial biomass, wide distribution and a broad ecological amplitude Bidwell et al., 2002, Xue et al., 2004  The terms phytoremediation and phytoextraction are sometimes incorrectly used as synonyms, but phytoremediation is a concept, whereas phytoextraction is a specific clean-up technology (Prasad and Freitas, 2003). Certain plants, called hyperaccumulators, absorb unusually large amounts of metals compared to other plants and the ambient metals concentration. Natural metal hyperaccumulators are plants that can accumulate and tolerate greater metal concentrations in shoots than those usually found in non-accumulators, without visible symptoms. Examples of commonly reported hyperaccumulators are given in Tables 2.7(a) and (b). According to Baker and Brooks (1989), hyperaccumulators should have a metal accumulation exceeding a threshold value of shoot metal concentration of 1% (Zn, Mn), 0.1% (Ni, Co, Cr, Cu, Pb and Al), 0.01% (Cd and Se) or 0.001% (Hg) of the dry weight shoot biomass. Over 400 hyperaccumulator plants have been reported, including members of the Asteraceae, Brassicaceae, Caryophyllaceae, Cyperaceae, Cunouniaceae, Fabaceae, Flacourtiaceae, Lamiaceae, Poaceae, Violaceae, and Euphobiaceae. Recently Environment Canada has released a database "Phytorem" which contains a worldwide inventory of more than 750 terrestrial and aquatic plants, both wild and cultivated species and varieties, of potential value for phytoremediation.  These plants are selected and planted at a site based on the metals present and site conditions. After they have grown for several weeks or months, the plants are harvested. Planting and harvesting may be repeated to reduce contaminant levels to allowable limits (Kumar et al., 1995). The time required for remediation depends on the type and extent of metal contamination,  33 the duration of the growing season, and the efficiency of metal removal by plants, but it normally ranges from 1 to 20 years (Kumar et al., 1995; Blaylock and Huang, 2000). This technique is suitable for remediating large areas of land contaminated at shallow depths with low to moderate levels of metal-contaminants (Kumar et al., 1995; Blaylock and Huang, 2000).  2.2.4.1 Types of phytoextraction  Two basic strategies of phytoextraction are being developed: chelate-assisted phytoextraction, which we term induced phytoextraction; and long-term continuous phytoextraction. If metal availability is not adequate for sufficient plant uptake, chelates or acidifying agents may be added to the soil to liberate them (Cunningham and Ow, 1996; Huang et al., 1997; Lasat et al., 1998). However, side effects of the addition of chelate to the soil microbial community are usually neglected. It has been reported (Wu et al., 1999) that many synthetic chelators capable of inducing phytoextraction might form chemically and microbiologically stable complexes with metals, threatening soil quality and groundwater contamination. Several chelating agents, such as EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycol-O,O'-bis-[2-amino-ethyl]- N,N,N',N',-tetra acetic acid), EDDHA (ethylenediamine di o-hyroxyphenylacetic acid), EDDS (ethylene diamine disuccinate) and citric acid, have been found to enhance phytoextraction by mobilizing metals and increasing metal accumulation (Tandy et al., 2006; Cooper et al., 1999). The increase in the phytoextraction of Pb by shoots of Zea mays L. was more pronounced than the increase of Pb in the soil solution with combined application of EDTA and EDDS (Luo et al., 2006). Although EDTA was, in general, more effective in soil metal solubilization, EDDS, less harmful to the environment, was more efficient in inducing metal accumulation in Brachiaria decumbens shoots (Santos, et al., 2006). However, there is a potential risk of leaching of metals to groundwater, and a lack of reported detailed studies regarding the persistence of metal-chelating agent complexes in contaminated soils (Lombi et al., 2001a,b).  2.2.4.2 Successful factors for phytoextraction of heavy metals  As a plant-based technology, the success of phytoextraction is inherently dependent on several plant characteristics, the two most important being the ability to accumulate large quantities of biomass rapidly and the capacity to accumulate large quantities of environmentally important metals in the shoot tissue (Kumar et al., 1995; Cunningham and Ow, 1996; McGrath, 1998,  34 Pilon-Smits, 2005). Effective phytoextraction requires both plant genetic ability and the development of optimal agronomic practices, including (i) soil management practices to improve the efficiency of phytoextraction, and (ii) crop management practices to develop a commercial cropping system. Ebbs et al. (1997) reported that B. juncea, while having one-third the concentration of Zn in its tissue, is more effective at removing Zn from soil than Thlaspi caerulescens, a known hyperaccumulator of Zn. The advantage is due primarily to the fact that Brassica juncea produces ten-times more biomass than Thlaspi caerulescens. Plants for phytoextraction should be able to grow outside their area of collection, have profuse root systems and be able to transport metals to their shoots. They should have high metal tolerance, be able to accumulate several metals in large amounts, exhibit high biomass production and fast growth, resist diseases and pests, and be unattractive to animals, minimizing the risk of transferring metals to higher trophic levels of the terrestrial food chain (Thangavel and Subhuram, 2004). Phytoextraction is applicable only to sites containing low to moderate levels of metal pollution, because plant growth is not sustained in heavily polluted soils. The land should be relatively free of obstacles, such as fallen trees or boulders, and have an acceptable topography to allow normal cultivation practices, utilizing agricultural equipment. Selected plants should be easy to establish and care for, grow quickly, have dense canopies and root systems, and be tolerant of metal contaminants and other site conditions which may limit plant growth.  Basic et al. (2006a,b) investigated the parameters influencing the Cd concentration in plants, as well as the biological implications of Cd hyperaccumulation in nine natural populations of Thlaspi caerulescens. Cd concentrations in the plant were positively correlated with plant Zn, Fe and Cu concentrations. The physiological and/or molecular mechanisms for uptake, transport and/or accumulation of these four metals interact with each other. They specified a measure of Cd hyperaccumulation capacity by populations and showed that Thlaspi caerulescens plants originating from populations with high Cd hyperaccumulation capacity had better growth, by developing more and bigger leaves, taller stems, and produced more fruits and heavier seeds. Liu et al. (2006) conducted a survey of Mn mine tailing soils and eight plants growing on Mn mine tailings. The concentrations of soil Mn, Pb, and Cd and the metal-enrichment traits of these eight plants were analyzed. It was found that Poa pratensis, Gnaphalium affine, Pteris vittata, Conyza canadensis and Phytolacca acinosa possessed specially good metal-enrichment and metal-  35 tolerant traits. In spite of the high concentration of Mn in P. pratensis, its lifecycle was too short, and its shoots were too difficult to collect for it to be suitable for soil remediation.  The effectiveness of phytoextraction of heavy metals in soils also depends on the availability of metals for plant uptake (Li et al., 2000). The rates of redistribution of metals and their binding intensity are affected by the metal species, loading levels, aging and soil properties (Han et al., 2003). Generally, the solubility of metal fractions is in the order: exchangeable > carbonate specifically adsorbed > Fe-Mn oxide > organic > residual (Li and Thornton, 2001). Ammonium nutrition of higher plants results in rhizosphere acidification due to proton excretion by root cells. Ammonium-fed sunflowers induced a strong acidification of the solution and, compared to the nitrate-fed sunflowers, a small modification in mineral nutrition and different Cd partitioning between root and shoot. Moreover, ammonium nutrition was found to induce a great mobilisation of a sparingly soluble form of cadmium (CdCO3) (Zaccheo et al., 2006). A lipid- transfer protein isolated from a domestic cultivar of barley grain, Hordeum vulgare has the affinity to bind Co (II) and Pb (II), but not Cd (II), Cu (II), Zn (II) or Cr (III). This suggests a new possible role of barley lipid-transfer protein for phytoextraction (Gorjanovic et al., 2006).  The slow desorption of metals in soils has been a major impediment to the successful phytoextraction of metal contaminated sites. Except for Hg, metal uptake into roots occurs from the aqueous phase. In soil, easily mobile metals such as Zn and Cd occur primarily as soluble or exchangeable, readily bioavailable form. Cu mainly occurs as organic complexes and Mo predominate in inorganically bound and exchangeable fractions. Slightly mobile metals such as Ni and Cr are mainly bound in silicates (residual fraction). Soluble, exchangeable and chelated species of trace elements are the most mobile components in soils, facilitating their migration and phytoavailability (Williams et al., 2006). Other species such as Pb occur as insoluble precipitates (phosphates, carbonates and hydroxyl-oxides) which are largely unavailable for plant uptake (Pitchel et al., 1999). Understanding the mechanisms of rhizosphere interaction, uptake, transport and sequestration of metals in hyperaccumulator plants will lead to designing novel transgenic plants with improved remediation traits (Eapen and Souza, 2005). Moreover, the selection and testing of multiple hyperaccumulator plants could enhance the rate of phytoremediation, giving this process a promise one for bioremediation of environmental contamination (Suresh and Ravishankar, 2004). Some of the recent reports on phytoextraction are summarized in Table 2.8.  36 Table 2.8. Recent reports on phytoextraction  Metal Plant studied Method of Phytoremediation Results Reference Cd, Zn Thlaspi caerulescens PE-C Physiological and molecular mechanisms for uptake, transport and accumulation of four heavy metals Cd, Fe, Cu and Zn interact with each other. T. caerulescens plants originating from populations with high Cd hyperaccumulation capacity had better growth. Revegetation of metal polluted soils with T. caerulescens could help activate their biochemical and microbial functionality. Different soils had various responses to acidification. A different optimum pH may exist for phytoextraction. Basic et al., 2006a; Basic et al., 2006b; Keller et al.,2006; Hammer et al.,2006; Hernandez- Allica et al. (2006);.Wang et al. (2006) Mn Gnaphalium affine D. Don Conyza canadensis (L.) Cronq PE-C G. affine and C. canadensis had excessive accumulation of Mn and could be useful in phytoremediation. The perennial herb Phytolacca acinosa Roxb. (Phytolaccaceae), which occurs in Southern China, was found to be a new manganese hyperaccumulator. Liu et al, 2006; Xue et al., 2004 Cu Elsholtzia splendens, and Trifolium repens PE-CA Application of glucose or citric acid significantly increased the extractable Cu concentration in planted and unplanted soils. Concentrations of Cu in the shoots of E. splendens were 2.6, 1.9 and 2.9 times of those of T. repens under no chelate, citric acid and glucose treatments, respectively.  Chen et al., 2006 Pb, As, Pb, Cu, Zn, Cd. Carrot, Lettuce and Tomato. Euphorbia,Verbascum and Astragalus. PE-C Except for carrot roots concentration less than ICP-OES detectable limits. Plants with high metal intake abilities escalate mobility of metals and increase contaminations on surface and subsurface. Pendergrass and Butcher (2006). Sagiroglu et al. (2006)  37 Metal Plant studied Method of Phytoremediation Results Reference Cu, Zn, Pb Sunflower PE-CA Synthetic chelating agents did not increase the uptake of metals for equal soluble concentrations in the presence and absence of chelates. Proper use of soil amendments increased the phytoextraction of Zn, Cu, Pb, Cd from contaminated soils. Tandy et al., 2006; Clemente et al., 2006; Chen et al., 2006 Cu and Fe Athyrium vokoscense PE and PM 1 g Cu and 0.1 g Fe recovered from 500 g soil. Removal rates of Cu and Fe in the contaminated soil were 82 and 95 % respectively. Application of (NLMWOA (Natural Low Molecular Weight Organic Acids) increased the extraction of Cu, with no enhancement of lead phytoextraction. Kobayashi et al., 2006; Evangelou et al., 2006 Se Astragalus bisulcatus and Brassica juncea PE There was a substantial improvement in Se accumulation (4 to 9 times increase) in transgenic plants. LeDuc et al., 2006 Cd Brassica napus and Brassica juncea PE Lipid changes in B. juncea, the well- known Cd-hyperaccumulator species, revealed greater stability of its cellular membranes to cadmium-stress compared to a Cd- sensitive specie, B. napus. An increase in cadmium content varying from 16 to 74%, compared to the non-inoculated control, was observed in rape plants cultivated in soil treated with 100 mg Cd kg−1 (as CdCl2) and inoculated with the cadmium-resistance bacterial strains from heavy metal- polluted soils. Quartacci et al., 2006; Belimov et al., 2005; Nouairi et al., 2006; Sheng and Xia, 2006  PE- PhytoExtraction, CA- Chelate Assisted, C- Continuous, PM- Phytomining   38 Phytoremediation has been combined with electrokinetic remediation, applying a constant voltage of 30 V across the soil. The combination of both techniques could represent a very promising approach to the decontamination of metal-polluted soils (O'Connor et al., 2003).  2.3 Handling of hazardous plant biomass after phytoremediation  Phytoextraction involves repeated cropping of plants in contaminated soil until the metal concentration drops to an acceptable level. Each crop is removed from the site. This leads to accumulation of huge quantities of hazardous biomass, which must be stored or disposed appropriately to minimize environmental risk. After harvesting, the methods of disposal of contaminated plants include approved secure landfills, surface impoundments, deep well injection, ocean dumping or incineration. The waste volume can be reduced by thermal, microbial, physical or chemical means. In one study, the dry weight of Brassica juncea for induced phytoextraction of lead amounted to 6 tons/ha containing 10,000 to 15,000 mg/kg metal on a dry weight basis (Blaylock et al., 1997). Composting and compaction can provide post- harvest treatment (Raskin et al., 1997 and Kumar et al., 1995).  Even though composting can significantly reduce the volume of the harvested biomass, metal- contaminated biomass still requires treatment prior to disposal. In the case of compaction, care should be taken to collect and dispose of the leachate. A conventional and promising route to utilize biomass produced by phytoremediation is through thermo-chemical conversion processes such as combustion, gasification and pyrolysis.  If phytoextraction could be combined with biomass generation and its commercial utilization as an energy source, then it could be turned into a profitable operation, with the residual ash available to be used as an ore (Brooks, 1998; Comis, 1996; Cunningham and Ow, 1996). Phytomining includes the generation of revenue by extracting soluble metals produced by the plant biomass ash, also known as bio-ore. With some metals like Ni, Zn, Cu, etc., the value of reclaimed metal may provide an additional incentive for phytoremediation (Chaney et al.1997, Watanabe 1997, Thangavel and Subhuram 2004).  39 2.4. Conclusions  Phytoremediation is still in its research and development phase, with many technical issues needing to be addressed. The results, though encouraging, suggest that further development is needed. Phytoremediation is an interdisciplinary technology that can benefit from many different approaches. Results already obtained have indicated that some plants can be effective in toxic metal remediation. The processes that affect metal availability, metal uptake, translocation, chelation, degradation, and volatilization need to be investigated in detail. Better knowledge of these biochemical mechanisms may lead to: (1) Identification of novel genes and the subsequent development of transgenic plants with superior remediation capacities; (2) Better understanding of the ecological interactions involved (e.g. plant–microbe interactions); (3) Appreciation of the effect of the remediation process on ecological interactions; and (4) Knowledge of the entry and movement of the pollutant in the ecosystem. In addition to being desirable from a fundamental biological perspective, findings will help improve risk assessment during the design of remediation plans, as well as alleviation of risks associated with the remediation. It is important that public awareness of this technology be considered, with clear and precise information made available to the general public to enhance its acceptability as a global sustainable technology. So far, most phytoremediation experiments have taken place on a laboratory scale, with plants grown in hydroponic settings fed metal diets. Both agronomic management practices and plant genetic abilities need to be optimized to develop commercially useful practice.  40 2.5 References  Abia, A. A., Horsfall, M., and Didi, O. (2003). The use of chemically modified and unmodified cassava waste for the removal of Cd, Cu and Zn ions from aqueous solution. Bioresource Technology, 90, 345–348.  Adriano, D. C., Wenzel, W. W., Vangronsveld, J. and Bolan, N. S. (2004). Role of assisted natural remediation in environmental cleanup. Geoderma, 122, 121-142.  Albasel, N. and Cottenie, A. (1985). Heavy metal contamination near major highways, industrial and urban areas in Belgium grassland. Water, Air, and Soil Pollution, 24, 103-109.  Al-Chalabi, A. S. and Hawker, D. (2000). Distribution of vehicular lead in roadside soils of major roads of Brisbane, Australia. Water, Air, and Soil Pollution, 118, 299-310.  Alloway, B. J. (1995). Soil processes and the behaviour of metals. In: Alloway BJ (ed) Heavy metals in soils. Blackie Academic and Professional, London, pp. 38–57  Alkorta, I., Herna´ndez-Allica, J., Becerril, J. M., Amezaga, I., Albizu, I. and Garbisu, C. (2004). Recent findings on the phytoremediation of soils contaminated with environmentally toxic heavy metals and metalloids such as zinc, cadmium, lead, and arsenic. Reviews in Environmental Science and Bio/Technology, 3, 71–90  Atkinson, R., Aschmann, S. M., Hasegawa, D., Eagle-Thompson, E. T. and Frankenberger, J. R. (1990). Kinetics of the atmospherically important reactions of dimethylselenide. Environmental Science and Technology, 24, 1326-1332.  Azaizeh, H. A., Gowthaman, S. and Terry, N. (1997) Microbial selenium volatilization in rhizosphere and bulk soils from a constructed wetland. Journal of Environmental Quality, 26(3), 666- 672.  Baker, A. J. M. and Brooks, R. R. (1989). Terrestrial higher plants which hyper accumulate metallic elements- review of their distribution, ecology, and phytochemistry. Biorecovery, 1, 81-126.  Baker, A. J. M., Reeves, R. D. and McGrath, S. P. (1991). In situ decontamination of heavy metal polluted soils using crops of metal accumulating plants-a feasibility study. In In-situ bioremediation, eds. R. E. Hinchee and R. F. Olfenbuttel, Butterworth-Heinemann, Stoneham, M. A, pp. 539-544.  Baker, A. J. M. and Walker, P. L. (1990). Ecophysiology of metal uptake by tolerant plants. Shaw, A. J., ed., Eds.; Heavy Metal Tolerance in Plants: Evolutionary Aspects. Boca Raton, FL, USA: 155-177.  Banuelos, G. S., Cardon, G., Mackey, B., Ben-Asher, J., Wu, L. P., Beuselinck, P., Akohoue, S. and Zambrzuski, S. (1993). Boron and selenium removal in B-laden soils by four sprinkler irrigated plant species. Journal of Environmental Quality, 22(4), 786-797.  41 Basic, N., Keller, C., Fontanillas, P., Vittoz, P., Besnard, G. and Galland, N. (2006a). Cadmium hyperaccumulation and reproductive traits in natural Thlaspi caerulescens populations. Plant Biology, 8, 64–72.  Basic, N., Salamin, N., Keller, C., Galland, N. and Besnard, G. (2006b). Cadmium accumulation and genetic differentiation of Thlaspi caerulescens populations. Biochemical Systematics and Ecology, 34(9), 667-677.  Baudouin, C., Charveron, M., Tarrouse, R. and Gall, Y. (2002). Environmental pollutants and skin cancer. Cell Biology and Toxicology, 18, 341–348.  Beath, O. A., Eppsom, H. F. and Gilbert, G. S. (1937). Selenium distribution in and seasonal variation of vegetation type occurring on seleniferous soils. Journal of the American Pharmaceutical Association, 26, 394–405.  Belimov, A. A., Hontzeas, N., Safronova, V. I., Demchinskaya, S. V., Piluzza G., Bullitta, S. and Glick, B. R. (2005). Cadmium-tolerant plant growth-promoting bacteria associated with the roots of Indian mustard (Brassica juncea L. Czern.). Soil Biology & Biochemistry, 37, 241–250.  Berti, W. R. and Cunningham, S. D. (2000). Phytostabilization of metals. In: I. Raskin and B.D. Ensley eds. Phytoremediation of toxic metals: using plants to clean-up the environment. New York, John Wiley & Sons, Inc., pp. 71-88.  Bidwell S. D., Woodrow, I. E., Batianoff, G. N. and Sommer-Knudsen, J. (2002). Hyperaccumulation of manganese in the rainforest tree Austromyrtus bidwillii (Myrtaceae) from Queensland, Australia. Functional Plant Biology, 29, 899-905.  Birch, G. E. and Scollen, A. (2003). Heavy metals in road dust, gully pots and parkland soils in a highly urbanised sub-catchment of Port Jackson, Australia. Australian Journal of Soil Research, 41, 1329-1342.  Blaylock, M. J. and Huang, J. W. (2000). Phytoextraction of metals. In: I. Raskin and B.D. Ensley eds. Phytoremediation of toxic metals: using plants to clean-up the environment. New York, John Wiley & Sons, Inc., pp. 53-70.  Blaylock, M. J., Salt, D. E., Dushenkov, S., Zakharova, O., Gussman, C., Kapulnik, Y., Ensley, B. D. and Raskin, I. (1997). Enhanced accumulation of Pb in Indian mustard by soil- applied chelating agents. Environmental Science and Technology, 31(3), 860-865.  Bolan, N. S., Adriano, D. C. and Naidu, R. (2003). Role of phosphorus in (im)mobilization and bioavailability of heavy metals in the soil-plant system. Reviews of Environmental Contamination and Toxicology, 177, 1–44.  Boonyapookana, B., Parkplan, P., Techapinyawat, S., DeLaune, R. D. and Jugsujinda, A. (2005). Phytoaccumulation of lead by sunflower (Helianthus annuus), tobacco (Nicotiana tabacum), and vetiver (Vetiveria zizanioides). Journal of Environmental Science and Health A, 40, 117-137.   42 Boularbah, A., Schwartz, C., Bitton, G., Aboudrar, W., Ouhammou, A., and Morel, J. L. (2006). Heavy metal contamination from mining sites in South Morocco: 2. Assessment of metal accumulation and toxicity in plants. Chemosphere, 63(5), 811-817.  Broadhurst, C. L., Chaney, R. L., Angle, J. S., Maugel, T. K., Erbe, E. F. and Murphy, C. A. (2004). Simultaneous hyperaccumulation of nickel, manganese, and calcium in Alyssum leaf trichomes. Environmental Science and Technology, 38, 5797-5802.  Brooks, R. R. (ed) (1998). Plants that hyperaccumulate heavy metals. Wallingford, CAB International. p. 384.  Caille, N., Swanwick, S., Zhao, F. J. and McGrath, S. P. (2004). Arsenic hyperaccumulation by Pteris vittata from arsenic contaminated soils and the effect of liming and phosphate fertilisation. Environmental Pollution, 132, 113-120.  Chandra, S. K., Kamala, C. T., Chary, N. S., Balaram, V. and Garcia, G. (2005). Potential of Hemidesmus indicus for phytoextraction of lead from industrially contaminated soils. Chemosphere, 58, 507-514.  Chaney, R. L. (1983). Plant uptake of inorganic waste constitutes. In: Parr, J.F., Marsh, P.B. and Kla, J.M. eds. Land treatment of hazardous wastes. Park Ridge, NJ, Noyes Data Corp., pp. 50-76.  Chaney, R. L., Malik, M., Li, Y. M., Brown, S. L., Brewer, E. P., Angle, J. S. and Baker, A. J. M. (1997). Phytoremediation of Soil Metals. Current Opinion in Biotechnology, 8, 279- 283  Chaudhry, T. M., Hayes, W. J., Khan, A. G. and Khoo, C. S. (1998). Phytoremediation— Focusing on accumulator plants that remediate metal-contaminated soils. Australian Journal of Ecotoxicology, 4, 37–51.  Chen, Y. X., Wang, Y. P., Wu, W. X.; Lin, Q. and Xue, S. G.(2006). Impacts of chelate-assisted phytoremediation on microbial community composition in the rhizosphere of a copper accumulator and non-accumulator. Science of the Total Environment, 356 (1-3), 247-255.  Clemente, R., Walker, J. D., Roig, A. and Bernal, P. M. (2003). Heavy metal bioavailability in a soil affected by mineral sulphides contamination following the mine spillage at Aznalc´ollar (Spain). Biodegradation, 14, 199–205.  Clemente, R., Almela, C. and Bernal, P. M. (2006). A remediation strategy based on active phytoremediation followed by natural attenuation in a soil contaminated by pyrite waste Environmental Pollution, 143(3), 397-406.  Comis, D. (1996). Green remediation: Using plants to clean the soil. Journal of Soil and Water Conservation, 51(3), 184-187.  Conesa, M. H., Faz, A., and Arnaldos, R. (2006). Initial studies for the phytostabilization of a mine tailing from the Cartagena-La Union Mining District (SE Spain). Chemosphere, 66(1), 38-44.  43 Cooper, E. M., Sims, J. T., Cunningham, S. D., Huang, J. W. and Berti, W. R. (1999). Chelate- assisted phytoextraction of lead from contaminated soil. Journal of Environmental Quality, 28, 1709–1719.  Cunningham, S. D. and Ow, D. W. (1996). Promises and prospects of phytoremediation. Plant Physiology, 110(3), 715-719.  Cunningham, S. D., Shann, J. R., Crowley, D. E. and Anderson, T. A. (1997). Phytoremediation of contaminated water and soil. In: Kruger, E.L.; Anderson, T.A. and Coats, J.R. eds. Phytoremediation of soil and water contaminants. ACS symposium series 664. Washington, DC, American Chemical Society, pp. 2-19.  Davis, T. A., Volesky, B., and Vieira, R. H. S. F. (2000). Sargassum seaweed as biosorbent for heavy metals. Water Research, 34, 4270–4278.  Dierberg, F. E., Débuts, T. A. and Goulet, J. R. N. A. (1987). Removal of copper and lead using a thin-film technique. In: Reddy, K.R. and Smith.W.H. eds. Aquatic plants for water treatment and resource recovery. Magnolia Publishing, pp. 497-504.  Desouza, M. P.; Pilon-Smits, E. A. H. and Terry, N. (2000). The physiology and biochemistry of selenium volatilization by plants. In: Raskin, I. and Ensley, B.D. eds. Phytoremediation of toxic metals: using plants to clean-up the environment. New York, John Wiley & Sons, Inc., pp. 171-190.  Dushenkov, V., Kumar, P. B. A. N., Motto, H. and Raskin, I. (1995). Rhizofiltration: the use of plants to remove heavy metals from aqueous streams. Environmental Science and Technology, 29, 1239-1245.  Dushenkov, S. and Kapulnik, Y. (2000). Phytofilitration of metals. In: Raskin, I. and Ensley, B.D. eds. Phytoremediation of toxic metals - using plants to clean-up the environment. New York, John Wiley & Sons, Inc., pp. 89-106.  Dushenkov, S., Vasudev, D., Kapulnik, Y.; Gleba, D., Fleisher, D., Ting, K. C. and Ensley, B. (1997a). Removal of uranium from water using terrestrial plants. Environmental Science and Technology, 31(12), 3468-3474.  Dushenkov, S., Vasudev, D., Kapulnik, Y., Gleba, D., Fleisher, D., Ting, K. C. and Ensley, B. (1997b). Phytoremediation: A novel approach to an old problem. In: WISE, D.L. ed. Global environmental biotechnology. Amsterdam, Elsevier Science B.V, pp. 563-572.  Eapen, S. and D'Souza, S.F. (2005). Prospects of genetic engineering of plants for phytoremediation of toxic metals. Biotechnology Advances, 23, 97-114.  Ebbs, S. D., Lasat, M. M., Brandy, D. J., Cornish, J., Gordon, R. and Kochian, L. V.(1997). Heavy metals in the environment: Phytoextraction of cadmium and zinc from a contaminated soil. Journal of Environmental Quality, 26, 1424-1430.  Elless, P. M., Poynton, Y. C., Williams, A. C., Doyle, P. M., Lopez, C. A., Sokkary, A. D., Ferguson, W. B. and Blaylock, J. M. (2005). Water Research, 39(16), 3863-3872.  44 Entry, J. A., Watrud, L. S. and Reeves, M. (1999). Accumulation of 137Cs and 90Sr from contaminated soil by three grass species inoculated with mycorrhizal fungi. Environmental Pollution, 104, 449-457.  Ensley, B. D. (2000). Rationale for use of phytoremediation. In: Raskin, I. and Ensley, B.D. eds. Phytoremediation of toxic metals: using plants to clean- up the environment. New York, John Wiley & Sons, Inc., pp. 3-12.  Evangelou, M. W. H., Ebel, M. and Schaeffer, A. (2006). Evaluation of the effect of small organic acids on phytoextraction of Cu and Pb from soil with tobacco Nicotiana tabacum. Chemosphere, 63 (6), 996-1004.  Evans, C. S., Asher, C. and Johnson, C. M. (1968). Isolation of dimethyl diselenide and other volatile selenium compounds from Astragalus racemosus (Pursh.) Australian Journal of Biological Sciences, 21, 13-20.  Fakayode, S. O and Olu-Owolabi, B. I. (2003). Heavy metal contamination of roadside topsoil in Osogbo, Nigeria: its relationship to traffic density and proximity to highways. Environmental Geology, 44(2), 150-157.  Fatoki, O. S. (1996). Trace zinc and copper concentration in roadside surface soils and vegetation: A measurement of local atmospheric pollution in Alice, South Africa. Environmental Interpretation, 22, 759-762.  Flathman, P. E. and Lanza, G. R. (1998). Phytoremediation: current views on an emerging green technology. Journal of Soil Contamination, 7(4), 415-432.  Frérot, H., Lefèbvre, C., Gruber, W., Collin, C., Dos Santos, A. and Escarre, J. (2006). Specific interactions between local metallicolous plants improve the phytostabilization of mine soils. Plant and Soil, 282, 53–65.  García, R. and Millán, E. (1998). Assessment of Cd, Pb and Zn contamination in roadside soils and grasses from Gipuzkoa (Spain). Chemosphere, 37, 1615- 1625.  Garbisu, C. and Alkorta, I. (2001). Phytoextraction: a cost-effective plant-based technology for the removal of metals from the environment. Bioresource Technology, 77, 229–236.  Gardea-Torresdey, J. L., de la Rosa, G. and Peralta-Videa, J. R. (2004). Use of phytofiltration technologies in the removal of heavy metals: A review. Pure and Applied Chemistry, 76(4), 801–813.  Ghosh, M. and Singh, S. P. (2005). A review on phytoremediation of heavy metals and utilization of its by-products. Applied Ecology and Environmental Research, 3(1), 1-18.  Glass, D. J. (1999). U.S. and international markets for phytoremediation, 1999-2000. Needham, Mass., D. Glass Associates, pp. 266.   45 Gorjanovic, S., Suznjevic, D., Beljanski, M. and Hranisavljevic, J. (2006). Barley lipid-transfer protein as heavy metal scavenger. Environmental Chemistry Letters, 2 (3), 113-116.  Gulson, B. L., Tiller, K. G., Mizon, K. J. and Merry, R. H. (1981). Use of lead isotopes in soils to identify the source of lead contamination near Adelaide, South Australia. American Chemical Society, 15(6), 691-696.  Han, F. X., Banin, A., Kingery, W. L., Triplrtt, G. B., Zhou, L. X., Zheng, S. J. and Ding, W. X. (2003). New approach to studies of heavy metal redistribution in soil. Advances in Environmental Research, 8, 113-120.  Hamme,r D., Keller, C., McLaughlin, M. J. and Hamon, R. E. (2006). Fixation of metals in soil constituents and potential remobilization by hyperaccumulating and non- hyperaccumulating plants: results from an isotopic dilution study. Environmental Pollution, 143(3), 407-415.  Heaton, A. C. P., Rugh, C. L., Wang, N. and Meagher, R. B. (1998). Phytoremediation of mercury - and methyl mercury - polluted soils using genetically engineered plants. Journal of Soil Contamination, 74, 497-510.  Hernandez-Allica, J., Becerril, J. M., Zarate, O. and Garbisu, C. (2006). Assessment of the efficiency of a metal phytoextraction process with biological indicators of soil health. Plant and Soil, 281 (1-2), 147-158.  Ho, Y B. and Tai, K. M. (1988). Elevated levels of lead and other metals in roadside soil and grass and their use to monitor aerial metal depositions in Hong Kong. Environmental Pollution, 49(1), 37-51.  Horsfall, M. and Abia, A. A. (2003). Sorption of cadmium (II) and zinc (II) ions from aqueous solutions by cassava waste biomass (Manihot sculenta Cranz). Water Research, 37, 4913– 4923.  Huang, J. W., Chen, J., Berti, W. R. and Cunningham, S. D. (1997). Phytoremediation of lead contaminated soil: role of synthetic chelates in lead phytoextraction. Environmental Science and Technology, 31(3), 800-805.  Hughes, J. B., Shanks, J., Vanderford, M., Lauritzen, J. and Bhadra, R. (1997) Transformation of TNT by aquatic plants and plant tissue cultures. Environmental Science and Technology, 31, 266-271.  Jain, S. K., Vasudevan, P., Jha, N. K. (1989). Removal of some heavy metals from polluted water by aquatic plants: studies on duckweed and water velvet. Biological Wastes, 28(2), 115-26.  Jaffre, T., Brooks, R. R., Lee, J. and Reeves, R. D. (1976). Sebertia acumip. A nickel- accumulating plant from new Caledonia. Science, 193, 579–580.  Kay, S. H., Haller, W. T. and Garrard, L. A. (1984). Effect of heavy metals on water hyacinths [Eichhornia crassipes (Mart.) Solms]. Aquatic Toxicology, 5, 117-128.  46  Keller, C., Diallo, S., Cosio, C., Basic, N., and Galland, N. (2006). Cadmium tolerance and hyperaccumulation by Thlaspi caerulescens populations grown in hydroponics are related to plant uptake characteristics in the field. Functional Plant Biology, 33(7), 673–684.  Knasmuller, S., Gottmann, E., Steinkellner, H., Fomin, A., Pickl, C., Paschke, A., God, R. and Kundi, M. (1998). Detection of genotoxic effects of heavy metal contaminated soils with plant bioassays. Mutation Research, 420, 37–48.  Kobayashi, F., Asada, C., Nakamura, Y. (2005). Phytoremediation of soil contaminated with heavy metals and recovery of valuable metals. Kagaku Kogaku Ronbunshu, 31 (6), 476- 480.  Kubota, H. and Takenaka, C. (2003). Arabis gemmifera is a hyperaccumulator of Cd and Zn. International Journal of Phytoremediation, 5, 197-120.  Kumar, P. B. A. N., Dushenkov, V., Motto, H. and Raskin, I. (1995). Phytoextraction: The use of plants to remove heavy metals from soils. Environmental Science and Technology, 29(5), 1232-1238.  Kuo, S., Jellum, E. J. and Baker, A. S. (1985). Effects of soil type, liming, and sludge application on zinc and cadmium availability to Swiss chard. Soil Science, 139, 122–130.  Lasat, M. M., Fuhrmann, M., Ebbs, S. D., Cornish, J. E. and Kochian, L. V. (1998). Phytoremediation of a radio cesium contaminated soil: evaluation of cesium- 137 bioaccumulation in the shoots of three plant species. Journal of Environmental Quality, 27(1), 165-168.  Lasat, M. M. (2002). Phytoextraction of toxic metals-A review of biological mechanisms. Journal of Environmental Quality, 31, 109-120.  Leblanc, M., Petit, D., Deram, A., Robinson, B., and Brooks, R. R. (1999). The phytomining and environmental significance of hyperaccumulation of thallium by Iberis intermedia from southern France. Economic Geology, 94(1), 109-113.  LeDuc, D. L., Samie, M. A., Bayon, M. M., Wu, C. P., Reisinger, S. J. and Terry, N. (2006). Overexpressing both ATP sulfurylase and selenocysteine methyltransferase enhances selenium phytoremediation traits in Indian mustard. Environmental Pollution, 144(1), 70- 76.  Lewis, B. G., Johnson, C. M. and Delwiche, C. C. (1966). Release of volatile selenium compounds by plants: collection procedures and preliminary observations. Journal of Agricultural and Food Chemistry, 14, 638-640.   47 Li, X. D. and Thornton, I. (2001). Chemical partitioning of trace and major elements in soils contaminated by mining and smelting activities. Applied Geochemistry, 16, 1693-1706.  Li, Y. M., Chaney, R. L., Angle, J. S and Baker, A. J. M. (2000). Phytoremediation of heavy metal contaminated soils in Bioremediation of contaminated soils. eds D.L Wise et al. Marcel Dekker, New York, pp. 837-884.  Liu, Y. G., Zhang, H. Z., Zeng, G. M., Huang, B. R. and Li, X. (2006). Heavy metal accumulation in plants on Mn mine tailings. Pedosphere, 16 (1), 131-136.  Lombi, E., Zhao, F. J., Dunham, S. J. and MacGrath, S. P. (2001a) .Phytoremediation of heavy metal-contaminated soils: Natural hyperaccumulation versus chemically enhanced phytoextraction. Journal of Environmental Quality, 30, 1919-1926.  Lombi, E., Zhao, F. J., Dunham. S. J. and McGrath, S. P (2001b). Cadmium accumulation in populations of Thlaspi caerulescens and Thlaspi geosingense. New Phytologist, 145, 11– 20.  Luo, C. L., Shen, Z. G., Li, X. D. and Baker, A. J. M. (2006). Enhanced phytoextraction of Pb and other metals from artificially contaminated soils through the combined application of EDTA and EDDS. Chemosphere, 63 (10), 1773-1784.  Mains, D., Craw, D., Rufaut, C. G. and Smith, C. M. S. (2006a). Phytostabilization of gold mine tailings, New Zealand. Part 1: Plant establishment in alkaline saline substrate. International Journal of Phytoremediation, 8 (2), 131-147.  Mains, D., Craw, D., Rufaut, C. G. and Smith, C. M. S. (2006b). Phytostabilization of gold mine tailings from New Zealand. Part 2: Experimental evaluation of arsenic mobilization during revegetation. International Journal of Phytoremediation, 8 (2), 163-183.  McEldowney, S., Hardman, D. J. and Waite, S. (1993). Treatment technologies. In Pollution, Ecology and Biotreatment pp. 48-58 eds. S. McEldowney, D.J. Hardman and S. Waite, Longman Singapore Publishers Pvt. Ltd. Singapore.  McGrath, S. P. (1998). Phytoextraction for soil remediation. In: Brooks, R.R., ed. Plants that hyperaccumulate heavy metals: their role in phytoremediation, microbiology, archaeology, mineral exploration and phytomining. New York, CAB International, pp. 261-288.  McGrath, S. P., Zhao, F. J. and Lombi, E. (2002). Phytoremediation of metals, metalloids, and radionuclides. Advances in Agronomy, 75, 1–56.  McGrath, S. P. and Zhao, F. J. (2003). Phytoextraction of metals and metalloids. Current Opinion in Biotechnology, 14, 277–282.  McIntyre, T. (2003). Phytoremediation of heavy metals from soils. Advances in Biochemical Engineering, Biotechnology, 78, 97–123.   48 Meagher, R. B. (2000). Phytoremediation of toxic elemental and organic pollutants. Current Opinion in Plant Biology, 3, 153–162.  Mkandawire, M. and Dudel, E. G. (2005). Accumulation of arsenic in Lemna gibba L. (duckweed) in tailing waters of two abandoned uranium mining sites in Saxony, Germany. Science of the Total Environment, 336, 81-89.  Mo, S. C., Choi, D. S. and Robinson, J. W. (1989). Uptake of mercury from aqueous solution by duckweed: the effect of pH, copper, and humic acid. Journal of Environmental Health, 24, 135-146.  Navari-Izzo, F. and Quartacci, M. F. (2001). Phytoremediation of metals- Tolerance mechanisms against oxidative stress. Minerva Biotecnologica,13, 73–83.  Nouairi, I., Ben Ammar, W., Ben Youssef, N., Daoud, D. B., Ghorbal, M. H. and Zarrouk, M.(2006). Comparative study of cadmium effects on membrane lipid composition of Brassica juncea and Brassica napus leaves. Plant Science, 170 (3), 511-519.  Odjegba, V. J. and Fasidi, I. O. (2004). Accumulation of trace elements by Pistia stratiotes: implications for phytoremediation. Ecotoxicology, 13, 637-646.  O'Connor, C. S., Leppi, N. W., Edwards, R. and Sunderland, G. (2003). The combined use of electrokinetic remediation and phytoremediation to decontaminate metal-polluted soils: a laboratory-scale feasibility study. Environmental Monitoring and Assessment, 84, 141– 158.  Parker, D. R., Feist, L. J., Varvel, T. W., Thomason, D. N. and Zhang, Y. Q. (2003). Selenium phytoremediation potential of Stanleya pinnata. Plant and Soil, 249, 157-165.  Pendergrass, A. and Butcher, D. J. (2006). Uptake of lead and arsenic in food plants grown in contaminated soil from Barber Orchard, NC. Microchemical Journal, 83(1), 14-16.  Pilon-Smits, E. A. H., Desouza, M. P., Hong, G., Amini, A., Bravo, R. C., Payabyab, S. T. and Terry, N. (1999). Selenium volatilization and accumulation by twenty aquatic plant species. Journal of Environmental Quality, 28(3), 1011-1017.  Pilon-Smits, E. A. H. (2005). Phytoremediation. Annual Review of Plant Biology, 56, 15-39.  Pitchel, J., Kuroiwa, K. and Sawyer, H . T. (1999). Distribution of Pb, Cd and Ba in soils and plants of two contaminated soils. Environmental Pollution, 110, 171-178.  Prasad, M. N. V and Freitas, H. (2003). Metal hyperaccumulation in plants - Biodiversity prospecting for phytoremediation technology. Electronic Journal of Biotechnology, 6, 275- 321.  Pugh, R. E., Dick, D. G. and Fredeen, A. L. (2002). Heavy metal (Pb, Zn, Cd, Fe and Cu) contents of plant foliage near the Anvil range lead/zinc mine, Faro, Yukon Territory. Ecotoxicology and Environmental Safety, 52, 273–279.   49 Quartacci, M. F., Argilla, A., Baker, A. J. M. and Navari-Izzo, F. (2006). Phytoextraction of metals from a multiple contaminated soil by Indian mustard. Chemosphere, 63 (6), 918- 925.  Raskin, I., Smith, R. D. and Salt, D. E. (1997). Phytoremediation of metals: using plants to remove pollutants from the environment. Current Opinion in Biotechnology, 8, 221-226.  Reeves, R. D. and Brooks, R. R. (1983). Hyperaccumulation of lead and zinc by two metallophytes from a mining area of Central Europe. Environmental Pollution Series A, 31, 277-287.  Rizzi, L., Petruzzelli, G., Poggio, G. and Vigna, G. (2004). Soil physical changes and plant availability of Zn and Pb in a treatability test of phytostabilization. Chemosphere, 57(9), 1039-1046.  Robinson, B. H., Brooks, R. R., Howes, A.W., Kirkman, J. H. and Gregg, P. E. H. (1997). The potential of the high-biomass nickel hyperaccumulator Berkheya coddii for phytoremediation and phytomining. Journal of Geochemical Exploration, 60, 115- 126.  Sagiroglu, A., Sasmaz, A and Sen, O. (2006). Hyperaccumulator plants of the Keban mining district and their possible impact on the environment. Polish Journal of Environmental Studies, 15 (2), 317-325.  Salt, D. E., Blaylock, M., Kumar, P. B. A. N., Dushenkov, V., Ensley, B. D., Chet, L. and Raskin, L. (1995). Phyto-remediation: a novel strategy for the removal of toxic metals from the environment using plants. Biogeochemistry, 13, 468-474.  Salt, D. E., Smith, R. D. and Raskin, I. (1998). Phytoremediation. Annual Review of Plant Physiology and Plant Molecular Biology, 49, 643–668.  Sánchez Martín, M.  J., Sánchez Camazano, M. and Lorenzo, L .F. ( 2000). Cadmium and lead contents in suburban and urban soils from two medium-sized cities of Spain: influence of traffic intensity. Bulletin of Environmental Contamination and Toxicology, 64, 250–257.  Santos, F. S., Hernández-Allica, J., Becerril, J. M., Amaral-Sobrinho, N., Mazur, N. and Garbisu, C. (2006). Chelate-induced phytoextraction of metal polluted soils with Brachiaria decumbens. Chemosphere, 65(1), 43-50.  Schnoor, J. L. (2000). Phytostabilization of metals using hybrid poplar trees. In: Raskin, I. and Ensley, B.D., eds. Phytoremediation of toxic metals: using plants to clean-up the environment. New York, John Wiley & Sons, Inc., pp. 133- 150.  Schwartz, C., Echevarria, G. and Morel, J. L. (2003). Phytoextraction of cadmium with Thlaspi caerulescens. Plant Soil, 24, 27-35.  Sekhar, K. C., Kamala, C. T., Chary, N. S., Sastry, A. R. K., Rao, T. N., and Vairamani, M. (2004). Removal of lead from aqueous solutions using an immobilized biomaterial derived from a plant biomass. Journal of Hazardous Materials, 108, 111–117.  50  Sharma, N. C., Gardea-Torresdey, J. L., Parsons, J. and Sahi, S. V. (2004). Chemical speciation and cellular deposition of lead in Sesbania drummondii. Environmental Toxicology and Chemistry, 23: 2068-2073.  Sheng, X. F., and Xia, J. J. (2006). Improvement of rape (Brassica napus) plant growth and cadmium uptake by cadmium-resistant bacteria. Chemosphere, 64(6), 1036-1042.  Singh, O. V., Labana, S., Pandey, G., Budhiraja, R. and Jain, R. K. (2003). Phytoremediation: an overview of metallic ion decontamination from soil. Applied Microbiology and Biotechnology, 61, 405–412.  Smith, R. A. H. and Bradshaw, A. D. (1992). Stabilization of toxic mine wastes by the use of tolerant plant populations. Transactions of the Institution of Mining and Metallurgy, 81, A230-A237.  Smolders, E. and Degryse, F. (2002). Fate and effect of zinc from tire debris in soil. Environmental Science and Technology, 36, 3706-3710.  Stoltz, E. and Greger, M. (2002). Accumulation properties of As, Cd, Cu, Pb and Zn by four wetland plant species growing on submerged mine tailings. Environmental and Experimental Botany, 47(3), 271-280.  Stoltz, E. (2004). Phytostabilisation:use of wet plants to treat mine tailings. Doctoral thesis, Department of Botany, Stockholm University.  Suresh, B. and Ravishankar, G. A. (2004). Phytoremediation - A novel and promising approach for environmental clean-up. Critical Reviews in Biotechnology, 24, 97-124.  Suszcynsky, E. M. and Shann, J. R. (1995). Phytotoxicity and accumulation of mercury subjected to different exposure routes. Environmental Toxicology and Chemistry, 14, 61- 67.  Sutherland, R. A., Day, J. P. and Bussen, J. O. (2003). Lead concentrations, isotope ratios and source apportionment in road deposited sediments, Honolulu, Oahu, Hawaii. Water, Air, and Soil Pollution, 142, 165-186.  Swaileh, K. M., Hussen, R. H. and Abu-Elhaj, S. (2004). Assessment of heavy metal contamination in road side surface soil and vegetation from the West Bank. Arch. Environmental Contamination and Toxicology, 47, 23-30.  Tandy, S., Schulin, R. and Nowack, B. (2006). The influence of EDDS on the uptake of heavy metals in hydroponically grown sunflowers. Chemosphere, 62(9), 1454-1463.  Terry, N., Carlson, C., Raab, T. K. and Zayed, A. (1992). Rates of selenium volatilization among crop species. Journal of Environmental Quality, 21, 341-344.  51  Thangavel, P. and Subhuram, C. V. (2004). Phytoextraction - Role of hyper accumulators in metal contaminated soils. Proceedings of the Indian National Science Academy. Part B, 70 (1), 109-130.  Tian, J. L., Zhu, H. T., Yang, Y. A. and He, Y. K. (2004). Organic mercury tolerance, absorption and transformation in Spartina plants. Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao (Journal of Plant Physiology and Molecular Biology), 30, 577-582.  Tordoff, G. M., Baker, A. J. M. and Willis, A. J. (2000). Current approaches to the revegetation and reclamation of metalliferous mine wastes. Chemosphere, 41(1-2), 219-228.  Turer,D., Maynard, J. B. and Sansalone, J. J. (2001). Heavy metal contamination in soils of urban Highways: Comparison between runoff and soil concentrations at Cincinnati, Ohio. Water, Air, and Soil Pollution, 132, 293–314.  Utsunamyia, T. (1980). Japanese Patent Application No. 55- 72959.  Viard, B., Pihan, F., Promeyrat, S. and Pihan, J. C. (2004). Integrated assessment of heavy metal (Pb, Zn, Cd) highway pollution: bioaccumulation in soil, Graminaceae and land snails. Chemosphere, 55(10), 1349-1359  Viklander, M. (1998). Particle size distribution and metal content in street sediments. Journal of Environmental Engineering, 124, 761-766.  Watanabe, M. E. (1997). Phyto-remediation on the brink of commercialization. Environmental Science and Technology, 31, 182-186.  Wang, J., Zhao, F., Meharg, A. A., Raab, A., Feldmann, J.,and McGrath, P .S (2002). Mechanisms of Arsenic Hyperaccumulation in Pteris vittata. Uptake Kinetics, Interactions with Phosphate, and Arsenic Speciation. Plant Physiology, 130, 1552-1561.  Wang, A. S., Angle, J. S., Chaney, R. L., Delorme, T. A. and Reeves, R. D. (2006). Soil pH effects on uptake of Cd and Zn by Thlaspi caerulescens. Plant and Soil, 281 (1-2), 325-337.  Wei, S. H., Zhou, Q. X., Wang, X., Cao, W., Ren, L. P. and Song, Y. F. (2004). Potential of weed species applied to remediation of soils contaminated with heavy metals. Journal of Environmental Science, (China) 16, 868-873.  Wenzel, W. W., Adriano, D. C., Salt, D. and Smith, R. (1999). Phytoremediation: A plant- microbe-based remediation system. In: SSSA (Ed), Bioremediation of Contaminated Soils Agronomy Monograph no. 37, SSSA, Madison, WI, USA, pp. 457–508.  WHO (1997). Health and Environment in Sustainable Development. WHO, Geneva.  52  Williamson, A. and Johnson, M. S. (1981). Reclamation of metalliferous mine wastes. In: Lepp, N.W. (Ed.), Effect of Heavy Metal Pollution on Plants, vol. 2. Applied Science Publishers, Barking, Essex, UK, pp. 185–212.  Williams, A. C., Nascimento, W., Amarasiriwardena, D. and Xing, B. (2006). Comparison of natural organic acids and synthetic chelates at enhancing phytoextraction of metals from a multi-metal contaminated soil. Environmental Pollution, 140(1), 114-123.  Wu, J., Hsu, F. C. and Cuningham, S. D. (1999). Chelate assisted Pb phytoextraction: Pb availability, uptake, and translocation constraints. Environmental Science and Technology, 33(11), 1898-1904.  Xue, S. G., Chen, Y. X., Reeves, R. D., Baker, A. J., Lin, Q. and Fernando, D. R. (2004). Manganese uptake and accumulation by the hyperaccumulator plant Phytolacca acinosa Roxb. (Phytolaccaceae). Environmental Pollution, 131, 393-399.  Yoon, J., Cao, X., Zhou, Q., and Ma, L .Q. (2006). Accumulation of Pb, Cu, and Zn in native plants growing on a contaminated Florida site. Science of the Total Environment, 368(2– 3), 456–464.  Zaccheo, P., Crippa, L. and Pasta, V. D. (2006). Ammonium nutrition as a strategy for cadmium mobilisation in the rhizosphere of sunflower. Plant and Soil, 283 (1-2), 43-56.  Zhu, Y. L., Zayed, A. M., Quian, J. H., De Souza, M. and Terry, N. (1999). Phytoaccumulation of trace elements by wetland plants: II Water hyacinth. Journal of Environmental Quality, 28, 339-344.  53 3. 2PRELIMINARY EXAMINATION OF FIELD MEASUREMENTS OF METAL ACCUMULATION - HEAVY METAL STATUS OF SOILS AND PLANTS PRIOR TO PHYTOREMEDIATION ALONG HIGHWAYS  3.1 Introduction  Road sediments typically contain elevated levels of contaminant metals, which may be mobilised by runoff waters (Sezgin et al., 2003). Motor vehicles constitute a principal source of these metals. Exposure to high levels of metals is linked to many adverse effects on human health and wild life. Phytoremediation can be successfully employed for the remediation of metal toxicity in soils (Chaney et al., 1997). Depending on the strategy adopted by plants, the remediation method can be either containment or removal (Thangavel and Subhuram, 2004). Containment by stabilisation may be suited for busy contaminated sites like highway soils where removal is neither feasible nor practical due to financial and other physical constraints. An evaluation of contaminant metal status and phytoremediation potential of local plants of the site is essential prior to an in situ soil phytoremediation program. Field measurements of metal accumulation (Cu, Pb, Mn and Zn) in soils and plants along highway sites and the potential for phytostabilisation of plant species were investigated. The selected sites for the present study (Appendix A, Figures 1 and 2) are located on the northern right of way of the Trans-Canada Highway (TCH) or HW1 close to the intersection with the 176th Street overpass in Surrey and northern ramp of HW 17, Deltaport Way, Delta, British Columbia.  Both highway sites have similar design i.e. elevated highway sections with overflow flush shoulder type of drainage, but with different surrounding geology, land use, average daily traffic, and predominant meteorological conditions. The overall objectives of this study were to charecterise the physico-chemical properties of highway soils, to characterise the extent and type of metal contamination (Pb, Zn, Cu and Mn) in these soils, to identify the plants that spontaneously colonise the polluted sites and to assess their metal accumulation capacities.  2  A version of this chapter has been published. Padmavathiamma, P.K, Li, L.Y. and Lavkulich, L. (2007) Heavy metal contamination and potential of local plants for phytoremediation along Highways. Biogeochemistry of Trace Elements: Environmental Protection, Remediation and Human Health, Edited: Y. Ahu, N. Lepp and R Naidu. pp.648-650.   54 Information obtained from this study provided insight for formulating the technical programs for subsequent studies to identify a package for remediating the metal-contaminated sites.  3.2 Materials and methods  3.2.1 Site description  The study was focused on two highway sites, Trans-Canada Highway (HW 1) at the 176th  Street intersection in Surrey, B.C and Highway 17 in Delta, B.C. Factors considered in site selection include: traffic characteristics, surrounding land use activities, meteorological conditions, pavement type and condition, drainage area, highway design characteristics, proximity to a receiving water body, highway maintenance practices, and logistical considerations (safety, accessibility, future development, etc.). For this study, both sites share most characteristics (except for traffic numbers, land use and meteorological conditions) and are thus similar enough to be suitable for comparison, but different enough to test the efficiency of the selected methodology.  Having two different study sites served two purposes: 1) to observe the influence of different geo-environmental conditions on soil metal loadings and plant accumulations, and 2) to assess the efficiency of the selected methodology for remediation in soils from two different road-side conditions.  3.2.1.1 Trans-Canada Highway & 176 th St. site description  This site is located on the northern right of way of the Trans-Canada Highway (TCH), close to the intersection with the 176 th Street overpass in Surrey, B.C. The highway corridor is oriented in a NW-SE direction and through the Port Mann bridge connects Surrey to Coquitlam and Port Coquitlam. The surface geology is classified as Capilano sediments, which are composed of marine to glaciomarine stony to stoneless silty loam to clayey loam with minor sand and silt. These sediments are normally less than 3 m thick, but in some areas may reach up to 30 m thick (Geological Survey of Canada,1998). Ministry of Transportation and Infrastructure records show traffic counts of 82,900 vehicles per day for the west-bound (WB) corridor and 73,100 for the  55 east-bound (EB). The surface comprises three lanes of asphalt pavement for each direction with a middle grassy area, flush shoulder type of surface drainage and a concrete barrier that separates on and off-ramps from the main corridor in the proximity of the 176 th street overpass. Construction of Highway 1 in this location was completed in the 1960s. Surrounding land use is a mixture of residential, agricultural, undeveloped and parkland. Characteristics of this study site are summarized in Appendix A, Table 1.  3.2.1.2 Highway 17 site  The study area is located on the northern ramp of HW 17, Deltaport way, Delta, B.C. At the study site location, this highway is oriented in a north-south direction. On a bigger scale Hwy 17 connects Tsawwassen and the ferry terminal to Highway 99, which in turn connects to the rest of the Lower Mainland. The area is located in the flat lowlands of the Fraser River delta. The underlying material is composed of soil deposits, primarily silts, clays and sands. These sediments were deposited over thousands of years by seasonal floodwaters that spread across these lowlands. They are important agricultural soils although in some cases poor drainage can be a problem. The BC Ministry of Environment, Lands & Parks has classified aquifers in the area as having moderate to high vulnerability to contamination, with low to moderate use (Geological Survey of Canada, 1998). The construction of Highway 17 was completed in the early 1970s. Surrounding land use in the area is agricultural. According to Ministry of Transportation and Infrastructure records, traffic counts for HW 17 are 20,417 and 22,899 vehicles/day for Northbound (NB) and Southbound (SB), respectively. The surface comprises two lanes in each direction made of asphalt pavement with flush shoulder surface drainage and a middle concrete barrier. Highway 17 characteristics are summarized in Appendix A, Table 2.  3.2.1.3 Background locations  It is important to distinguish between anthropogenic contamination and background or natural levels to enable accurate evaluation of the degree of contamination in an area. The background site for HW 1 is at the bak yard of Surrey fire hall, 1 km north of the main intersection, HW1 with 176 street in Surrey, B.C, whereas the background site for HW 17 is near the Boundary Bay airport, which is north west of the main HW 17 site. The soils in these regions have minimal  56 impacts from anthropogenic effects and have the same physico-chemical characteristics as that of main soils.  3.2.2 Collection of samples and laboratoratory analysis  Soil sampling was performed in transects normal to the road. Samples were collected at intervals of 1, 2, 4, 6 and 8 m from the side of the road and at depths of 0-15 and 15-30 cm. Past experiences from Ministry of Transportation and Infrastructure with roadside soils of B.C have shown that metal contamination from above ground sources is generally restricted to the surface layers (Preciado and Li, 2006). The rationale for sampling at different horizontal distances and depths was to assess the influence of traffic behaviour on the migration of metals in the soil. Vigorously growing plants in the study sites were also collected. The soil samples were air-dried for 5 days, and then sieved through a 2-mm mesh after crushing the clods gently using a wooden mallet. Plant samples were thoroughly washed with running tap water and rinsed with de-ionized water to remove soil/sediment particles attached to the plant surfaces. Shoots and roots were then separated and oven dried (70°C) to constant weight. The dried tissues were weighed and finely ground for the analysis of Cu, Pb, Mn and Zn. Basic characteristics of the soil such as pH, electrical conductivity, total carbon, texture, CEC were estimated (Table 3.1). The recoverable heavy metals in the soil sample were estimated by the method proposed by EPA (Smoley, 1992). After dry ashing the plant samples (Lintern et al., 1997), the ash was dissolved in 10 mL of 1 M HCl and diluted to 50 mL with de-ionized water. Both soil and plant extracts were analysed for metals using a Varian Spectre AA 220 Multi-element Fast Sequential Atomic Absorption Spectrometer. Statistical techniques were used to aid in the sampling and interpretation of results. The computer software ORIGIN along with EXCEL was used for statistical computations, including mean values, standard deviation (SD) and metal concentration ratios. To assess the accumulation characteristics and translocation properties of metals in plants, Enrichment Coefficient (EC) and Translocation Factor (TF) were determined.  ECroot   = [Metal]root/[Metal]soil  ECshoot = [Metal]shoot/[Metal]soil  TF = [Metal]shoot/[Metal]root    57 3.3 Results and discussion  The basic characteristics of two highway soils are given below (Table 3.1).  Table 3.1 Basic soil characteristics of Highway soils Soil characteristics    HW1 (main site) HW1 (BG site) HW 17 (mainsite) HW 17  (BG site) pH in water (Hendershot et al., 2008a) 5.51 5.42 5.60 5.69 Electrical Conductivity (Lavkulich, 1981) 1.10 (dS/m) 0.82 (dS/m) 0.73 (dS/m) 0.64 (dS/m) Total Carbon (Sheldrick, 1984) 2.10 % 1.50% 3.90 % 2.2 % Total Nitrogen 0.18 % 0.13% 0.30 % 0.19% Cation Exchange Capacity (Hendershot et al., 2008b) 19 molc kg-1 15 molc kg-1 22 molc kg-1 20 molc kg-1 Available P (Bray and Kurtz, 1945) 10.9 mg/kg 10.4 mg/kg 6.1 mg/kg 10.7 mg/kg Texture (Kettler et al., 2001) Sandy clay loam Sandy clay loam Silty clay loam Silty clay loam Soil classification Luvisolic humoferric podzol Luvisolic humoferric podzol Humic luvic gleysol.  Humic luvic gleysol.   Concentrations of Cu, Pb, Mn and Zn in soil from two highway sites were found to decrease with increasing distance from the road (Table 3.2 and Figure 3.1). Background (BG) concentrations are given in Table 3.3. The total metal concentrations in HW1 soil were found to be higher than for HW 17 soil, probably because of higher traffic for HW1 compared to HW 17. The decrease of the metal concentrations with distance from the road indicates that vehicular emissions play a significant role in determining the levels of heavy metals in the roadside soil. In the case of Pb, a rapid decline with distance was observed, probably the result of the highly  58 immobile nature of Pb. There have been reports of higher levels of Pb in top soil along major roads in several other cities and of correlations of the metals with traffic volume (e.g. Onianwa, 2001). With respect to Cu and Zn, even though a declining trend was observed with distance from the road, the variation was relatively modest. Typically, abrasions of motor vehicle parts and vehicle emissions, as well as geological parent material, have been demonstrated to be principal sources of these types of metals in road sediments throughout the world (Onianwa, 2001). The use of Tetraethyl lead (TEL) as an antiknock compound for gasoline engines during the early 1970s and subsequent replacement by methyl cyclopentadienyl manganese tricarbonyl (MMT) might have contributed to the high Pb and Mn concentrations in highway soils.  Table 3.2 Soil metal concentrations with distance from the highway (HW1). Mean values ± S.D, n = 3.  Table 3.3. Metal concentrations in background (BG) sites (0-15 cm) Background (BG) site Cu (mg/kg) Pb (mg/kg) Mn (mg/kg) Zn (mg/kg) HW1 (Fire hall soil) 52±5.7 93±11 215±5.9 70±11 HW17 (near Boundary Bay air port) 31±9 20±6 81±17 50±11 Mean values ± S.D, n = 3.    Depth (cm) 0-15 15-30 0-15 15-30 0-15 15-30 0-15 15-30 Metal concentrations (mg/kg) Distance from the Highway (m) Cu Pb Mn Zn 1 74±8.0 ±247 472±28 156±10 266±8.9 ±7.4341 129±5.1 ±4.595 2 47±4.6 ±3.655 264±33 162±4.0 374±4.8 ±4.6394 94±4.7 ±6.088 4 32±4.6 ±5.626 210±11 ±8.0103 278±10.8 ±13.7301 85±7.4 ±6.170 6 28±3.8 20±3.0 54±7.0 ±4.630 230±3.2 ±6.0270 81±5.3 ±5.064 8 25±3.3 ±3.718 28±8.0 ±2.918 218±3.4 ±6.6238 80±5.9 ±6.359  59  Figure 3.1 Soil metal concentrations with distance from the highway (0-15 cm), a comparison. (a) HW 1 (b) HW 17, (mean values ± SD, n = 3).  Plants were collected from the study site at two different times, first late in February (winter) and then towards the end of July (summer), 2006. The plants collected during February were Juncus effusus, Holcus lanatus, Festuca rubra and a moss, Rhytidiadelphus squarrosus. Those collected during the summer were Equisetum arvense, Rumex occidentalis, Plantago lanceolata, Ranunculus occidentalis and Rumex acetosella. All plants, except for weedy species were 0 100 200 300 400 500 600 1 2 4 6 8 Distance from the Highway (m) So il m et al  co n ce n tr at io n s (m g/ kg ) Cu Pb Mn Zn 0 100 200 300 400 500 600 1 2 4 6 8 Distance from the Highway (m) So il m et al  co n ce n tr at io n s (m g/ kg ) Cu Pb Mn Zn (a) HW 1 (b) HW 17  60 assessed for metal accumulation and translocation properties. The plants that spontaneously colonised the two sites are given in Appendix A, Figures 7 and 8. Metal concentrations in the plants seen at highway sites during both seasons (winter and summer) are given in Figure 3.2. In the case of Festuca rubra and Holcus lanatus, the concentration of metals in the roots was greater than that of the shoots, revealing a low translocation rate for metals. This indicates low mobility of metals from the roots to the shoots and immobilization of heavy metals in the roots, suggesting an exclusion strategy (Figure 3.2). The metal concentrations in Juncus effusus (both root and shoot) are found to be lower than those of Festuca rubra and Holcus lanatus and there is not a predominant difference between the root and shoot metal concentrations (Figure 3.2), indicating good translocation of metals to the above ground portions of the plant. 0 50 100 150 200 250 Root Shoot Root Shoot Root Shoot Root Shoot Cu Pb Mn Zn Pl a n t m e ta l c o n c e n tr a tio n s  (m g/ kg ) Juncus effusus Festuca rubra Holcus lanatus   Figure 3.2 Concentration of metals in the plants that spontaneously colonised the study sites (mean values±SD, n = 3). Since Festuca rubra and Holcus lanatus have a high root EC (Enrichment Coefficient) and low TF (Translocation Factor) with values <1, (Table 3.4), they have the potential for phytostabilization (Kumar et al., 1995). Because Holcus lanatus belongs to the weedy species, Festuca rubra together with other plants selected from Phytorem (Environment Canada, 2003) were tested in the later studies on phytostabilisation, which are explained in subsequent chapters. The moss (Rhytidiadelphus squarrosus) collected from the study site, was found to accumulate substantial quantities of Pb followed by Mn, Zn and Cu (Figure 3.3). Mosses do not have a developed root system and the metal accumulation may be directly from the atmosphere. Hence it can be used as a bio-indicator for atmospheric deposition of metals.  61 Table 3.4  Root/ Shoot ratio, Enrichment Coefficient (EC) and Translocation Factor (TF)  Cu Pb Mn Zn Plant species R/S ratio ECR ECS TF ECR ECS TF ECR ECS TF ECR ECS TF Festuca rubra 0.9- 1.2 1.2 0.98 0.43 0.63 0.47 0.74 0.93 0.87 0.93 1.27 0.85 0.82 Holcus lanatus 0.16- 0.31 1.1 0.94 0.63 0.51 0.39 0.76 0.50 0.43 0.85 1.50 1.60 0.85 Mean values, n = 3. R/S ratio – Root/Shoot ratio, ECR – Enrichment Coefficient, root, ECS - Enrichment Coefficient, shoot, TF – Translocation factor.   0 50 100 150 200 250 Cu Mn Pb Zn Metals M e ta l c o n c e n tr a tio n  (m g/ kg )  Figure 3.3 Metal concentrations (mg/kg) in moss (Rhytidiadelphus squarrosus), n = 3.  3.4 Conclusions  • Concentrations of Cu, Pb, Mn and Zn decreased with increasing distance from the highways.  • High values for metal accumulation in plants from highway soils were observed and variations among plant species were noticed for metal accumulation.  • Festuca rubra, which spontaneously colonizes the contaminated site, was found to be an ideal candidate for further phytostabilisation studies. The moss collected from the study site, Rhytidiadelphus squarrosus, was found to be a promising indicator for heavy metal accumulation, especially Pb. • The basic information obtained from this study provided insight to formulate the technical program for the subsequent studies.  62 3.5 References Bray, R. H. and Kurtz, L.T. (1945). Determination of total, organic and available forms of phosphorus in soils. Soil Sci. 59, 39-45.  Chaney, R .L., Malik, M., Li, Y. M., Brown, S. L., Brewer, E .P., Angle, J .S. and Baker, A .J. M. (1997). Phytoremediation of soil metals. Curr. opin. Biotechnol., 8, 279-283.  Hendershot, W. H., Lalande, H. and Duquette, M. (2008a). Soil reaction and exchangeable acidity in soil. In: Carter, M. R. and Gregorich, E. G., eds. Soil sampling and methods of analysis, second edition. CRC Press, Taylor &. Francis, Boca Raton. pp. 173-175.  Hendershot, W. H., Lalande, H. and Duquette, M. (2008b). Ion exchange and exchangeable cations. In: Carter, M. R. and Gregorich, E. G. eds. Soil sampling and methods of analysis, second edition. CRC Press, Taylor &. Francis, Boca Raton. pp. 203-205.  Kettler, T. A., Doran, J. W. and Gilbert, T. L. (2001). Simplified method for particle-size determination to accompany soil-quality analyses, Soil Sci. Am. J. 65, 849-852.  Kumar, P. B. A. N., Dushenkov, V., Motto, H. and Raskin, I. (1995). Phytoextraction: The use of plants to remove heavymetals from soils. Environ. Sci. Technol., 29(5), 1232-1 238.  Lavkulich, L. M. (1981). Methods manual, Pedology Laboratory. Department of Soil Science, University of British Columbia, Vancouver.  Lintern, M. J., Butt, C. R. M. and Scott, K. M. (1997). Gold in vegetation and soil-three case studies from the goldfields of southern Western Australia. J. Geochem. Explor., 58(1), 1- 14.  Onianwa, P. C. (2001). Roadside topsoil concentrations of lead and other heavy metals in Ibadan, Nigeria. Soil Sediment Contam., 10(6), 577 591.  Preciado, H. F. and Li, L. Y. 2006. Evaluation of metal loadings and bioavailability in air, water and soil along two Highways of British Columbia, Canada. Water, Air, and Soil Pollution, 172, 81–108.  Sheldrick, B. H. (1984). Total Carbon, LECO induction furnace. Analytical Methods Manual 1984. Research Branch, Agriculture Canada, Ottawa, ON.  Sezgin, N., Ozcan, H. K., Demir, G., Nemlioglu,S. and Bayat, C. (2003). Determination of Heavy Metal Concentrations in Street Dusts in IstanbulE-5 Highway. Environment International, 29, 979-985.  Smoley,C. K.(1992). Methods for the determination of metals in environmental samples. Environmental Monitoring systems Laboratory, U.S. E.P.A., Cincinnati, Ohio.  Thangavel, P. and Subhuram, C. V. (2004). Phytoextraction - Role of hyper accumulators in metal contaminated soils. Proc. Indian Natn. Sci. Acad., B, 70 (1), 109-130.  63  4. 3PHYTOREMEDIATION OF METAL-CONTAMINATED SOIL IN TEMPERATE HUMID REGIONS OF BRITISH COLUMBIA, CANADA  4.1 Introduction  Metal contamination in soils usually results from industrial activities, such as mining and smelting, electroplating, gas exhaust, energy and fuel production, fertilizer and pesticide application, and municipal waste (Kabata-Pendias and Pendias, 2001). The highway system is a potential source of metal-contaminants to the surrounding environment through natural mechanisms such as atmospheric dust deposition or through the hydrologic cycle (Preciado and Li, 2006). Storm water generated by runoff from roads and highways contains metals in toxic concentrations (Barrett et al., 1998; Characklis and Weisner, 1997; Legret and Pagotto, 1999), due to loading from various sources related to vehicles, road construction materials, and road management (Hallberg et al., 2007). High concentrations of metals, particularly lead, zinc, manganese, iron, and copper, in highway runoff result from the wear of brakes, tires and other vehicle parts, leakage of lubricants, exhaust emission etc. (Preciado and Li, 2006). Because these metals do not degrade naturally, their high concentrations in runoff in both particulate and dissolved forms can result in accumulation in roadside soil at levels that are toxic to organisms in surrounding environments (Sansalone and Buchberger, 1997). Exposure to high levels of these metals has been linked to adverse effects on human health and wildlife. Toxic metals damage DNA, and their carcinogenic effects in animals and humans are probably related to their mutagenic ability (Baudouin et al., 2002).  Several techniques have been developed to treat storm water runoff in highways. These include treating storm water runoff with a humic filter media, a pelletized compost medium capable of removing up to 82-98% of the metals (Richman, 1997), using straw coated with sulphide compounds to bind the metals (Robert et al., 2003), or a grassy swale to reduce the concentration of metals in the highway runoff (Barrett et al., 1998).  3  A version of this chapter has been published. Padmavathiamma, P.K. and Li, L.Y. (2009) Phytoremediation of metal-contaminated soil in temperate humid regions of British Columbia, Canada. International Journal of Phytoremediation, 11(6): 575-590.   64 Phytoremediation is an effective technique to remediate metals in soils (Salt et al., 1995; Chaney et al., 1997), since it represents a more sustainable, cost-effective and environmentally-friendly tool for cleaning metal-polluted soils than alternative remediation methods (Baker et al., 1994). Reducing the environmental impact by holding the metal-pollutants at the source location in non-mobile forms so that they do not interfere with the normal life processes of the vegetated cover is phytostabilisation (Smith and Bradshaw, 1979).  A typical right of way for roads in Canada is around 30 m, and at least 33% of that land in the right of way is unpaved and can support animal life, where metal contamination needs to be remediated. Phytostabilisation requires least maintenance among different phytoremediation techniques, and it could be a feasible and practical method of remediating in roadside soils along highways and for improving highway runoff drainage.  Although many plants have been reported to be effective for phytoremediation of metal- contaminated soil, there is a lack of research addressing the phytoremediation of roadside soils subjected to multi-component metal solutions, like those subjected to continuous atmospheric and highway runoff loadings. Many phytoremediation studies were for a single metal and determine the metal uptake by plant at one stage of growth (Hamlin and Parker, 2006; Weng et al., 2005; Meyers et al., 2008). To the best of our knowledge very little work has been done on the phytoremediation efficiencies of plants in a multi-metal-contaminated scenario. Investigating the effects of uptake pattern, biometric characters and biomass accumulation can contribute to the potential for phytoextraction and phytostabilisation.  The present study involved a systematic and comprehensive effort to assess the phytoremediation potential of five plant species, commonly available in regions with temperate maritime climate, for a highway soil in southwest British Columbia. The research protocol involved: (1) estimating the metal uptake by plants at different growth stages; (2) assessing the translocation properties and metal accumulation characteristics, then examining the relationship of bio-metrics and biomass of the plants with metal accumulations; and (3) determining the efficiencies of these plants for phytoextraction and phytostabilisation in soils with multiple metal contaminations. Comprehensive pot tests with randomized experimental design using five plant species and four metals commonly found in highway roadside soils (i.e. Cu, Pb, Mn and Zn) were carried out under outdoor conditions. Detailed analyses were carried out to characterize the  65 chemical and physiological aspects of the plants. The results are intended to contribute to identifying suitable plant species for remediation of metals along highways. This will aid in determining best management practices for phytoremediation of metal contamination in soils, due to traffic activities leading to air deposition and highway runoff.  4.2 Materials and methods  The soil used for this research was collected from the yard of Surrey Fire Hall No. 5, located 1 km north of the intersection of TCH (Trans Canada Highway) with the 176 Street overpass in Surrey, British Columbia (a busy site with respect to traffic counts, >80,000 vehicles/day). The sampling site has the same soil as the nearby highway intersection (Luttmerding, 1980). The metal concentrations of the soils studied are given in Table 4.1. The soil at this site had a layer of organic enriched debris, about 5 cm thick, which was removed prior to testing as it would not be typical of a highway soil, and the first 15 cm of soil was collected and brought to the laboratory for the experiments.  Table 4.1. Metal concentrations according to British Columbia CSR (Contaminated Sites Regulation) standards and studied soil metal concentrations in the pot study.  British Columbia CSR Standards Cu (mg/kg) Pb (mg/kg) Mn (mg/kg) Zn (mg/kg) A 30 50 200 80 B 100 500 1000 500 C 500 1000 2000 1500 Studied Soils B0 52 93 215 70 BA 80 146 408 148 BC 520 1100 2160 1600 Level A is the investigation standard for residential and recreational land use. Level B is the remediation standard for residential and recreational land use, and the investigation standard for commercial and industrial use. Level C is the remediation standard for exclusive commercial and industrial activities (Ministry of Environment, British Columbia, 1995).  4.2.1 Experimental details  The concentrations of metals studied were based on previous work (Fakayode and Olu-Owolabi, 2003; Preciado and Li, 2006) and the British Columbia Standards for contaminated sites (Ministry of Environment, British Columbia, 1995). Soils were studied with three different metal  66 concentrations: (a) B0  the original soil containing the following concentrations of the four metals which are the focus of this study: Cu 52 mg/kg, Pb 93 mg/kg, Mn 215 mg/kg, Zn 70 mg/kg. (b) BA  the original soil spiked with addition of all four metals to give total concentrations of Cu, Pb, Mn, and Zn of 80, 146, 408 and 148 mg/kg, respectively. (c) BC  the original soil spiked to provide total Cu, Pb, Mn, and Zn concentrations of 520, 1100, 2160, and 1600 mg/kg, respectively.  Table 4.1 summarizes these concentrations in comparison with the CSR (Contaminated Sites Regulation) standard levels. In the present study, the original soil (B0) was considered as the background soil, since it was collected about 1 km from the main highway intersection and had the same physico-chemical characteristics as the main highway soil. There was no soil that contained no metals in the present study. Hence, the results are compared among plants grown in soils with different metal contamination levels.  The outdoor pot experiments were conducted during May – September, 2006 in the Totem Field of the University of British Columbia, in a Completely Randomized Design with five plant species, three different concentrations of multi-metals and three replicates. The five plant species tested were Lolium perenne L (perennial rye grass), Festuca rubra L (red fescue), Helianthus annuus L (sunflower), Poa pratensis L (Kentucky bluegrass) and Brassica napus L (rape).  Forty-five plastic pots of diameter 150 mm and height 200 mm were used in each experiment. Two sets of 45 pots were set up for destructive sampling at two stages of plant growth. The weight of each pot without soil was determined before filling it with 1kg soil. The carrier salts for soil spiking with metals were CuSO4, Pb(C2H3O2)2, MnSO4  and ZnSO4. The soil in each pot was mixed with the required concentration of multi-metals in 400 mL of distilled water bringing up the moisture content to the field moisture capacity (36%). This was kept as such for two days for equilibration and seeds sown (0.5 g/pot). The pots were watered for the first two weeks with a complete nutrient solution of N, P, K, Ca and Mg (Hoagland and Arnon, 1950). When the plants were established (after 4 weeks), the pots were transferred to the outdoors to simulate field conditions and watered as needed. The summary of weather conditions during this p  Sampling was carried out at two stages of plant growth, 90 and 120 DAS (days after eriod (May 2006 to December 2006) is given in Appendix C, Table 2. sowing). Among the different plant  67 species, Brassica flowered first, so the samples were taken at its maximum flowering stage, 90 DAS and senescence stage, 120 DAS. Even though these stages were different for other species, for consistency and comparability, all species were sampled at the same time to assess their efficiencies for metal uptake. Hereafter, stages of sampling appear in the text, tables and figures as 90 DAS and 120 DAS.  4.2.2 Bio-metric observations  Several bio-metric characters such as root length, number of branches per root, shoot length, number of leaves per plant, shoot weight, root weight and root/shoot ratio were recorded at 90 and 120 DAS. Root length and shoot length were measured using a measuring tape.  4.2.3 Laboratory analysis  The plant samples were thoroughly washed with running tap water and rinsed with de-ionized water to remove any soil/sediment particles attached to the plant surfaces (Spirochova et al., 2003). Shoots and roots were then separated and oven dried (70ºC) to constant weight. The dried tissues were weighed and ground for analysis of Cu, Pb, Mn and Zn.  After dry ashing of plant samples (Lintern et al., 1997), the ash was dissolved in 10 mL 1 M HCl and diluted to 50 mL with de-ionized water. Plant extracts were analysed for Cu, Pb, Mn and Zn using a Varian Spectre AA 220 Multi-element Fast Sequential Atomic Absorption Spectrometer. Quality checks and control were performed using blanks, duplicate samples and reference materials.  4.2.4 Statistical analyses  The statistical significance of differences among means was determined by one-way analysis of variance (ANOVA) followed by least significant difference (LSD) tests. In order to assess the efficiency of plants for phytoextraction and phytostabilisation, the Enrichment Coefficient (EC) of root (Croots/Csoil = ratio of root concentration to soil concentration) and shoot (Cshoots/Csoil = ratio of shoot concentration to soil concentration) and Translocation Factor (TF = Cshoots/Croots = ratio of shoot concentration to root concentration) were calculated (Kumar et al., 1995 and Mattina et al., 2003). Correlation and regression analyses were conducted to establish the  68 relationship between different parameters. The strength of each relationship was interpreted according to the correlation classification of Hopkins (2000), namely negligible: 0.0–0.09; low: 0.1–0.29; moderate: 0.3–0.49; high: 0.5–0.69; very high: 0.7–0.89; nearly perfect: 0.9–1.0. When R was statistically significant at P ≤ 0.05, an asterisk (*) is provided to denote the statistical significance.  4.3 Results and discussion  The key characteristics of the studied soil are given in Table 4.2.  Table 4.2 Key characteristics of the original soil sample  Parameters Values Ratio of soil to gravel 1.9 pH in water 5.3 pH in 0.01M CaCl2 4.9 pH in 1M KCl 4.8 Electrical Conductivity 1.7 dS m-1 Total Carbon 1.4 % Total Nitrogen 0.15 % Cation Exchange Capacity 21 molc kg-1 Texture Sandy clay loam Soil classification Luvisolic humoferric podsol  The percentage germination of seeds in various treatments after 14 days of sowing is given in Figure 4.1. Poa had the highest germination, followed by Lolium, Brassica, Festuca, and Helianthus. There was no germination in BC soils, whereas the germination in B0 and BA soils was almost the same for all plant species. However, seedlings of Brassica (BrBA) and Helianthus (HBA)  did not establish in BA  soils, since the plants died one month after germination (i.e. ~45 days after sowing).   69 0 20 40 60 80 100 120 Lolium Festuca Helianthus Poa Brassica Plant species %  ge rm in a tio n B0 BA BC   Figure 4.1 Germination per cent (mean values). Error bars represent means ±S.D. for three replicates.  The difference may be due to the variation among the seeds of the plant species in tolerating different metal concentrations. Similar observations were reported by Adriano (2001) and Wong and Bradshaw (1982), where metal concentrations, even at low levels, can delay or prevent seed germination and establishment. The results discussed in this paper are for B0, BA, LB0  (Lolium B0 soil), LBA (Lolium BA soil), FB0 (Festuca B0 soil), FBA (Festuca BA soil), PB0 (Poa B0 soil), PBA (Poa BA soil), HB0 (Helianthus B0 soil) and BrB0 (Brassica B0 soil). Hereafter these abbreviations appear in the text, figures and tables.  4.3.1 Metal concentrations in plants  Two aspects are important in analyzing and interpreting metal accumulations in plants: (1) metal concentration, which is the amount of metal accumulating in plants per unit weight (i.e. mg/kg dry weight) (2) metal content or total metal uptake by plants in a pot, which indicates the metal removal /pot.  The metal concentrations in plants (mg/kg) at 120 DAS are given in Figure 4.2 (a) – (d). The plants grown in BA soil had metal concentrations, nearly twice for Cu and Pb, three times for Mn and four times for Zn compared to those grown in B0  soil. None of the plant species studied accumulated metals sufficiently to satisfy the criterion of a hyper-accumulator, as defined by Baker and Brooks (1989), i.e. a metal accumulation exceeding a threshold value of shoot metal concentration of 1% (Zn and Mn) and 0.1% (Cu and Pb) of the dry weight shoot biomass.    70  Root Shoot   Figure 4.2. Metal concentrations in plants at 120 DAS. (a) Cu, (b) Pb, (c) Mn, (d) Zn. (1. LB0, 2. LBA, 3. FB0, 4. FBA, 5. HB0, 6. PB0, 7. PBA, 8. BrB0). Error bars represent means ±S.D. for three replicates.  Plants with the highest metal concentrations, were Festuca for Cu (FB0 and FBA), Helianthus for Pb and Zn (HB0) and Poa for Mn (PB0 and PBA) as shown in Figure 4.2 (a), (b), (c) and (d) respectively. Poa (PBA) for Mn, differed significantly (P ≤ 0.05) from the other plant species in BA, whereas Festuca (FB0) for Cu had significant differences (P ≤ 0.05) compared to other plant species in B0 (Figure 4.2). Cu, Pb, Mn and Zn concentrations in plants at 120 DAS were higher than those at 90 DAS, and the root concentrations were higher than the shoot concentrations except in Poa for Mn and Helianthus and Brassica for Zn. There was an increase of 5 - 20% Cu, 2 - 10% Pb, 5 – 20% Mn and 11 – 15% Zn at 120 DAS compared to 90 DAS. Similar observations of high Cu accumulation in Helianthus roots were made by Lin et al. (2003) who found that nearly 60% of the total Cu in the roots of Helianthus annuus L. was bound to the cell-wall fraction and the plasma membrane. 0 20 40 60 80 100 120 1 2 3 4 5 6 7 8 Treatments Cu  co n ce n tr at io n  (m g/ kg ) 0 20 40 60 80 100 120 1 2 3 4 5 6 7 8 Treatments Pb  co n ce n tr at io n  (m g/ kg ) 0 200 400 600 800 1000 1200 1 2 3 4 5 6 7 8 Treatments M n  co n ce n tr at io n  (m g/ kg ) 0 100 200 300 400 500 600 1 2 3 4 5 6 7 8 Treatments Zn  co n ce n tr at io n  (m g/ kg ) (a) (b) (c) (d)  71 Cu concentrations reported by Stoltz and Greger (2002) and Shu et al. (2002) in wetland plant species and Paspalum distichum at maturity are higher than the values measured in the present study, possibly due to variations of the plant species and levels of Cu contamination of soils.  4.3.2 Metal content in plants  The metal content or the uptake by the plants (root, shoot and total) in a pot appears in Table 4.3. The metal uptake, which denotes the total metal extraction or removal by plant, is more important for assessing efficiencies of plants for phytoremediation than metal concentration in plant (mg/kg).  Lolium recorded a significantly higher uptake (P ≤ 0.05) for all metals at 120 DAS than the other plant species studied, which may be attributed to the high biomass (both root and shoot) exhibited by this species (Table 4.3). The metal uptake at 120 DAS was found to be significantly higher (P ≤ 0.05) than that at 90 DAS for both root and shoot. The metal uptake or removal by plants was found to increase with increasing soil metal loading (Table 4.3). This is mainly due to the high metal concentration in the tissues (Figure 4.2), since the biomass yields were similar for the two loadings (i.e. for B0 and BA soils).  Pb uptake by Helianthus and Brassica grown in B0 soil was higher than that by Lolium, Poa and Festuca grown in both B0  and BA soils at 90 DAS. This may be due to the differences among the plant species in Pb uptake. Also a high total Pb concentration in the soil does not necessarily result in high Pb concentrations in the plants due to the insoluble and immobile nature of Pb in soil (Blaylock et al., 1997). Helianthus has been reported to concentrate Pb in the leaf and stem indicating that it has the prerequisites of a hyper-accumulator plant (Boonyapookana et al., 2005). However in the present study, the concentration of Pb in Helianthus roots was higher than in shoots, both at 90 and 120 DAS, possibly due to the interactive effects of other metals. Even though the metal concentrations of plants in the present study were in the phyto-toxic range according to Levy et al. (1999), no marked visual changes in plant growth were observed.    72 Table 4.3. Metal uptake (µg/pot) by plants (roots, shoots and total) at 90 and 120 DAS  90 DAS 120 DAS  Treatments Root Shoot Total Root Shoot Total LB0 16.16 37.84 54.00b 74.10 89.60 163.70d LBA 22.44 82.10 104.53d 97.50 102.90 200.40e FB0 16.92 23.07 39.99a 48.60 34.80 83.40b FBA 24.00 31.11 55.11b 47.60 44.90 92.40b HB0 9.03 68.78 77.80c 15.60 85.00 100.60b PB0 10.02 32.07 42.08a 33.70 65.00 98.70b PBA 20.99 36.88 57.88b 52.29 83.30 135.59c Cu BrB0 8.22 39.39 47.61a 11.55 52.50 64.05a  LB0 20.69 20.3 40.99ab 97.47 50.68 148.15d LBA 23.00 52.29 77.99c 92.43 48.51 140.94d FB0 18.51 11.22 29.73a 49.32 16.62 65.94a FBA 22.97 11.93 34.90a 45.63 14.90 60.53a HB0 16.36 63.00 79.36c 23.32 57.75 81.07b PB0 14.04 17.22 31.26a 45.44 37.25 82.69b PBA 22.96 20.97 53.14b 60.42 41.82 102.24c Pb BrB0 16.89 37.81 66.10c 19.46 43.50 62.96a  LB0 91.68 236.60 328.28a 290.07 635.00 925.07b LBA 292.32 1207.50 1499.82b 988.00 1453.20 2441.20c FB0 75.96 88.04 164.00a 204.30 147.90 352.20a FBA 230.72 281.01 511.73a 427.00 387.09 814.09a HB0 62.35 510.00 572.35a 87.75 527.50 615.25a PB0 96.90 261.60 358.50a 296.40 618.80 915.20b PBA 286.20 746.12 1032.32b 700.52 1482.40 2182.92c Mn BrB0 81.94 402.80 484.74a 86.10 562.50 648.60a  LB0 46.56 117.60 164.16a 197.60 274.40 472.00b LBA 176.40 1035.30 1211.70e 683.80 932.40 1616.20d FB0 41.40 75.02 116.42a 109.80 103.53 213.33a FBA 144.96 210.12 355.08c 288.53 297.39 587.92b HB0 29.00 372.50 401.50c 45.63 402.50 448.13b PB0 44.20 118.81 163.01a 135.63 290.00 425.63b PBA 220.32 430.56 650.88d 430.77 817.70 1248.4d Zn BrB0 29.92 210.90 240.80b 346.50 282.50 629.00c Unit - µg/pot n = 3, F values significant at P < 0.05 for all metals. Means followed by a common letter in the same column for each metal do not differ significantly from each other according to the LSD test (P ≤ 0.05).    73 Correlations between metal concentrations in plants at 120 DAS (Table 4.4) revealed a positive interaction or a synergism between the studied metals. In the case of multi-metal contamination, soil analysis results can provide a measure of the ‘availability’ of each metal; but interactions between metals may occur, at the root surface affecting uptake, and within the plant affecting translocation and toxicity (Kabata-Pendias and Pendias, 2001). From the strengths of correlations obtained, it could be seen that maximum interaction was between Cu and Zn (R = 0.899), followed by Zn and Mn (R = 0.706), Pb and Zn (R = 0.618) and Pb and Cu (R = 0.557). According to the correlation classification of Hopkins (2000), correlations were very high for Cu & Zn and Zn & Mn, high for Cu & Pb and Zn & Pb and moderate for Mn & Pb and Cu & Mn (Table 4.4).  Table 4.4. Correlations between metal concentrations (mg/kg) in plants (shoot) at 120 DAS  Metals R Cu & Pb 0.557* Zn & Pb 0.618* Mn & Pb 0.467 Cu & Mn 0.458 Cu & Zn 0.899* Zn & Mn 0.706*          *Correlation coefficient was statistically significant at P ≤ 0.05.  The synergism between studied metals reveals that they are unlikely to compete with each other at the site of adsorption, absorption or translocation. This is likely due to low to moderate metal contamination levels in the present study. Further study on metal competition in plant uptake is necessary to confirm the findings in Table 4.4.  Thus based on metal concentration values expressed in mg/kg dry weight, Festuca had the highest accumulation for Cu, Helianthus for Pb and Zn and Poa for Mn. On the other hand,  74 based on uptake values (metal removal/pot), Lolium can be considered as the best candidate among the five studied plants for phytoextraction and Festuca for phytostabilisation. However, metal concentration ratios in plants and soil are the key characteristics that decide the suitability of plant species for phytoextraction/phytostabilisation.  4.3.3 Metal accumulation characteristics  The Enrichment Coefficient (EC) and Translocation Factor (TF) values (Kumar et al., 1995 and Mattina et al., 2003) help to identify the suitability of plants for phytoextraction and phytostabilisation. They explain the accumulation characteristics and translocation properties of metals in plants. Significantly higher values of ECshoot (P ≤ 0.05) at 120 DAS were found in Festuca (FB0) for Cu (1.98), Helianthus (HB0) for Pb (0.27) and Zn (5.90), and in Poa (PBA) for Mn (4.15) (Table 4.5).  The metal retention in roots, as revealed by ECroot at 120 DAS, was highest in Festuca (FB0) for Cu (2.79), Helianthus (HB0) for Pb (0.72), Poa (PBA) for Mn (4.01) and Lolium (LBA) for Zn (5.1) (Table 4.5). The ability of Poa to accumulate Mn was reported by Liu et al. (2006) in a previous study. Spirochova et al. (2003) reported more Pb retention in the roots of corn plants in a similar study. Pb retention in the root is based on binding to ion exchangeable sites on the cell wall and extracellular precipitation, mainly in the form of lead carbonates deposited in the cell wall (Dushenkov et al., 1995).  According to Kumar et al. (1995), a plant suitable for phytostabilisation should have higher EC for roots than for shoots and TF<1. The lowest TF values in the present study were recorded by Lolium for Cu and Pb (LBA at 90 and 120 DAS), Festuca for Mn (FB0 and FBA at 90 and 120 DAS) and Poa for Zn (PB0 and PBA at 90 and 120 DAS). Based on the EC and TF values, for phytoextraction, Festuca was found to be the best for Cu, Helianthus for Pb and Zn and Poa for Mn; whereas for phytostabilisation, Lolium was found to be the best for Cu and Pb, Festuca for Mn, and Poa for Zn.      75 Table 4.5. Translocation Factor (TF) and Enrichment Coefficient (EC) of metals.  Translocation factor Enrichment Coefficient 120 DAS 90 DAS 120 DAS 90 DAS Treatments     Shoot Root Shoot Root 0.82 0.79 1.71 2.08 1.12 1.40 LB0 0.65 0.69 1.18 1.81 0.66 1.10 LBA 0.83 0.80 1.98 2.79 1.48 1.88 FB0 0.73 0.81 1.60 1.90 1.40 1.96 FBA 0.84 0.88 1.65 1.99 1.30 1.48 HB0 0.97 0.99 1.61 1.66 1.27 1.28 PB0 0.77 0.73 1.07 1.78 0.78 1.06 PBA 0.66 0.85 1.04 1.12 0.94 1.12 BrB0  Cu * * ns * * ns F 0.35 0.33 0.23 0.53 0.17 0.50 LB0 0.30 0.31 0.18 0.55 0.16 0.51 LBA 0.34 0.35 0.22 0.63 0.20 0.57 FB0 0.37 0.41 0.20 0.54 0.19 0.46 FBA 0.40 0.45 0.27 0.72 0.29 0.64 HB0 0.32 0.38 0.17 0.54 0.17 0.46 PB0 0.32 0.35 0.18 0.55 0.15 0.47 PBA 0.33 0.41 0.21 0.67 0.22 0.55 BrB0 Pb ns * ns * ns ns F 1.40 0.88 1.70 1.20 1.24 1.40 LB0 0.91 0.82 3.10 3.40 1.87 2.30 LBA 0.75 0.67 1.31 1.75 1.02 1.50 FB0 0.80 0.76 2.64 3.30 2.05 2.69 FBA 0.93 0.94 1.63 1.74 1.45 1.50 HB0 0.86 0.84 2.11 2.40 1.80 2.14 PB0 1.03 1.02 4.15 4.01 3.20 3.12 PBA 0.91 0.87 1.64 1.80 1.49 1.69 BrB0 Mn    * * * * * * F 0.84 0.86     3.50 4.22 3.33 3.90 LB0 0.94 0.99 4.82 5.13 3.24 3.31 LBA 0.97 1.05 4.31 4.51 3.92 3.72 FB0 0.91 0.90 4.33 4.20 3.31 3.60 FBA 1.38 1.49 5.92 4.32 4.82 3.21 HB0 0.80 0.77 3.81 4.81 3.11 4.13 PB0 0.78 0.76 3.92 5.02 3.70 4.80 PBA 1.14 1.26 4.02 3.61 3.73 2.91 BrB0 Zn * * * * * * F F values compare the EC and TF values for different treatments (LB0, LBA, FB0, FBA, HB0, PB0, PBA, BrB0) at each stage, 90 DAS and 120 DAS. Mean values. n = 3. ns – F not significant, * - F significant at P ≤ 0.05.   76 Metal-tolerant species with high ECroot and low TF can be used for phytostabilisation of metal- contaminated sites (Yoon et al., 2006). The efficiencies of plants for phytostabilisation are determined by metal retention in roots since there is less translocation of metals to the above- ground portions. This enables harvestable biomass to continue growing uninhibited by metals and further reduces the passage of metals into the food chain through the activity of herbivores (McIntyre, 2003). In plants with TF < 1, there is less translocation of metals to the above-ground portions. The lower translocation of metals to the above-ground portions may be due to immobilization of metals in roots by vacuole sequestration or cell wall binding, thereby preventing interaction with high-molecular-weight compounds in the plant cell cytoplasm (Salt et al., 1995; Macfie and Welbourn, 2000). In the present study, TF was < 1 for all plant species except in Poa (PBA) for Mn and Helianthus (HB0) and Brassica (BrB0) for Zn.  It could be seen that for Lolium, when the soil Zn concentration was increased, more Zn was translocated from roots and accumulated in the tops of the plant (TF, 0.99 and 0.94 in LBA, compared to 0.86 and 0.84  in LB0 at 90 and 120 DAS). Zn accumulated in the shoot portion has been reported to be concentrated in chloroplasts, vacuole fluids and cell membranes (Kabata- Pendias and Pendias, 2001). The accumulation of Zn by the above-ground portions is representative of the Zn phytoextraction potential of plants (Hamlin and Parker 2006). Because of the high CEC of plant cell walls, metals are usually transported as chelated species rather than in ionic form. Hence the capacity of Zn to bind with organic anions is an important aspect that controls the plant transport of Zn.  Thus based on the results of the present study, Lolium, Poa and Festuca can be recommended for phytostabilisation of soils contaminated with moderate levels of Cu, Pb, Mn and Zn.  4.3.4 Relationships of metal concentration and bio-metric characters of plants  The biometric characteristics of plants and plant biomass are important parameters affecting plant health and successful functioning for metal remediation. Based on the correlation and regression analyses of each biometric character (i.e. number of leaves/plant, number of root branches, root length and shoot length) with plant metal concentrations, it is found that root  77 length, shoot length and number of branches/root are the main characteristics that influence the metal concentrations in plants.  Table 4.6. Correlations between metal concentrations (mg/kg) in plants and biometric characters at 120 DAS   Metal   Root length  Shoot length  No. of branches/root Cu Root RE  y = -4.4208x + 121.01  y = -0.8298x + 101.73  y = 0.5252x + 51.088   R  0.565*  -0.255  0.564*  Shoot RE  y = -3.618x + 78.014  y = -0.6625x + 61.682  y = 0.9447x + 10.998   R  0.509  -0.629*  0.518  Pb Root RE  y = -2.8878x + 91.446  y = -0.2825x + 70.192  y = 0.7057x + 3.8408   R  0.487  0.620*  0.708*  Shoot RE  = -0.6775x + 27.436  y = -0.0493x + 21.882  y = 0.9296x + 34.144   R  0.227  0.156  0.323  Mn Root RE  y = -72.053x + 1251.7  y = -13.133x + 1006.1  y = -6.9427x + 435.41   R  -0.599*  0.647*  -0.477  Shoot RE  y = -64.302x + 1093.7  y = -11.231x + 801.58  = -2.2643x + 265.9   R  -0.022  0.565*  -0.286  Zn Root RE  y = -50.455x + 838.21  y = -7.8505x + 564.26  y = -0.7197x + 170.77   R  -0.131  0.701*  0.189  Shoot RE  y = -45.199x + 757.61  y = -6.7467x + 509.92  y = 4.1943x + 66.159   R  -0.503  0.643*  0.3469  R - Correlation coefficient, RE - Regression Equation. * R was statistically significant at P ≤ 0.05.  78  Table 4.6 summarizes the relationship between metal concentrations and plant biometric characters at 120 DAS. Root parameters such as root length and number of branches/root have a significant influence (P ≤ 0.05) on root and shoot concentrations of Cu and Pb, whereas, Mn and Zn concentrations are significantly influenced by shoot length. In the case of Pb, a significant and positive correlation is obtained between root concentration and number of branches/root (R = 0.708).  Root parameters control the efficiency of rhizosphere metal dynamics and ultimately the efficiency of plants for soil metal de-contamination (Uren and Reisenauer, 1998; Marschner et al., 1995). The amount and composition of root exudates released into the rhizosphere are highly variable and dependent on plant species, stage of plant growth, physico-chemical environment and metal toxicity stress (Marschner et al., 1995). This accounts for the differential absorption of metals by plant species at different growth stages and different soil contamination levels in the present study.  4.3.5 Relationships of metal content and biomass of plants  The metal contents of plants per pot at 120 DAS are plotted against biomass levels in Figure 4.3. Significant and positive correlations (P ≤ 0.05) are obtained between the Pb content of roots and root biomass (R = 0.909), Pb content of shoots and shoot biomass (R = 0.902), Cu content of roots and root biomass (R = 0.814), Cu content of shoots and shoot biomass (R = 0.705) (Figure 4.3). Mn and Zn contents in plants have only nonsignificant correlations with the plant biomass for both root and shoot, and the values are not presented in Figure 4.3. The very low metal content in plants in the present study compared to many previous studies may be due to low biomass accumulation per pot. This could be due to the fact that, in this study, seeds were directly sown in the multi-metals contaminated soil and their survival ability and metal uptake pattern could have been affected as discussed above. Most previous studies on phytoremediation were conducted in hydroponic cultures (e.g. Tandy et al., 2006; January et al., 2008), or seeds were sown in potting mix or sand mix and the seedlings transplanted to the contaminated soils (e.g. Marchiol et al., 2004; Bhattacharya et al., 2006). Low levels of metal accumulation may also be due to interactions between metal ions in multi-metal-contaminated soils, inhibiting  79 metal uptake (Ebbs and Kochian, 1997), relative to soils having elevated levels of single metals (Huang and Cunningham, 1996; Blaylock et al., 1997).   Figure 4.3.  Relationship between metal content in plants and plant biomass (dry weight) at 120 DAS. (a) Root biomass and Cu content in roots. (b) Shoot biomass and Cu content in shoots. (c) Root biomass and Pb content in roots. (d) Shoot biomass and Pb content in shoots.  Such interactions could also explain why Brassica napus and Helianthus annuus accumulated less Pb and Zn in shoots than those reported by previous authors (Huang and Cunningham, 1996; Ebbs et al., 1997). In this study, Lolium recorded the highest values for shoot and root biomass (2.8 and 1.9 g/pot) at 120 DAS. However, Festuca recorded the highest root/shoot ratio, followed by Poa. Root biomass and root/shoot biomass ratio are important aspects of phytostabilisation, since the efficiency of rhizosphere metal precipitation depends on the exposure of plant roots to the contaminated zones.   Lolium, Poa and Festuca have been identified as being suitable for phytostabilisation of metal- contaminants (Cu, Pb, Mn and Zn) in highway soils. Growing these plant species along highway y = 28.674Ln(x) + 61.447 R = 0.814 0 20 40 60 80 100 120 0 0.5 1 1.5 2 2.5 Root  biomass (g/pot) Cu   co n te n t i n  ro o t (m ic ro  gr am s/ po t) y = 25.444Ln(x) + 59.131 R = 0.705 0 20 40 60 80 100 120 0 1 2 3 Shoot  biomass (g/pot) Cu   co n te n t i n  sh o o t (m ic ro  gr am s/ po t) y = 31.954Ln(x) + 69.598 R = 0.909 0 20 40 60 80 100 120 0 0.5 1 1.5 2 2.5 Root  biomass (g/pot) Pb  co n te n t i n  ro o t (m ic ro  gr am s/ po t) y = 20.989Ln(x) + 30.119 R = 0.902 0 10 20 30 40 50 60 70 0 1 2 3 Shoot  biomass (g/pot) Pb  co n te n t i n  sh o o t (m ic ro  gr am s/ po t)  80 soils could also help to reduce the metal concentration of highway runoff by filtering or trapping metal-containing particulates and reducing the amount of metal-contaminated sediments entering the biota. It is suggested that these plants be tested under field conditions.  4.4 Conclusions and recommendations  • The germination of seeds is affected by the metal concentrations. In BC soils (total Cu, Pb, Mn, and Zn concentrations of 520, 1100, 2160, and 1600 mg/kg, respectively), none of the seeds germinated because of metal toxicities.  • Plant metal concentrations depend on the original soil metal concentrations. The metal concentrations in plants increased by nearly a factor of 2 for Cu and Pb, three times for Mn and four times for Zn in BA soils, compared to B0  soils .   • The efficiency of plants to accumulate metals followed the order, Festuca > Lolium > Helianthus > Poa > Brassica for Cu, Helianthus > Brassica > Festuca > Poa > Lolium for Pb, Poa > Festuca > Lolium > Brassica > Helianthus for Mn and Helianthus > Festuca > Poa > Brassica > Lolium for Zn. There was an increase of 5 - 20% Cu, 2 - 10% Pb, 5 – 20% Mn and 11 – 15% Zn in plants at 120 DAS compared to 90 DAS.  • The plant biomass plays a significant role in total metal removal by plants. Lolium recorded a significantly higher uptake (P < 0.05) for all the metals than the other plant species studied, as Lolium had the highest biomass (both root and shoot), whereas Festuca recorded the lowest uptake for all four metals.  • Metal retention in roots, as revealed by the values of ECroot at 120 DAS, was highest in Festuca (FB0) for Cu, Helianthus (HB0) for Pb, Poa (PBA) for Mn and Lolium (LBA) for Zn. Based on the EC (Enrichment Coefficient) and TF (Translocation Factor) values, for phytoextraction, Festuca was found to be the best for Cu, Helianthus for Pb and Zn and Poa for Mn. For phytostabilisation, Lolium was found to be best for Cu and Pb, Festuca for Mn, and Poa for Zn.  • The effects of combined metals on plant metal uptake are complex, and further study is required.   81 • Lolium, Poa and Festuca are suggested for pilot study of highway roadside soil remediation to evaluate if they can mitigate the metal toxicity of highway runoff by sequestering metal contaminants in roots or rhizosphere. In the field, interactions of various pedogenic and biogenic factors need to be considered.  • Improvements to phytostabilisation could be obtained by species mixtures and implementing soil management practices that have a positive influence on the efficiency of this process. The presence and concentration of the appropriate transport proteins or translocating chelating molecules in plants play a very important role in the translocation of metals. These factors should be investigated in future studies.  82 4.5 References  Adriano, D. C. (2001). Trace Elements in Terrestrial Environments: Biogeochemistry, Bioavailability and Risks of Metals (Second edition), Springer-Verlag, New York.  Baker, A. J. M. and Brooks, R. R. (1989). Terrestrial higher plants which hyperaccumulate metallic elements—a review of their distribution, ecology and phytochemistry. Biorecovery, 1, 81–126.  Baker, A. J. M., McGrath, S. P., Sidoli, C. M. D. and Reeves, R. D. (1994). The possibility of in situ heavy metal decontamination of polluted soils using metal-accumulating plants. Resour. Conserv. Rec., 11, 41–49.  Barrett, M. E., Irish, L. B., Malina, J. F. and. Charbeneau, R. J. (1998). Characterization of highway runoff in Austin, Texas area. J. Environ. Eng., 124, 131–137.  Baudouin, C., Charveron, M., Tarrouse, R., and Gall, Y. (2002). Environmental pollutants and skin cancer. Cell Biology and Toxicology, 18, 341–348.  Bhattacharya, T., Banerjee, D. K. and Gopal, B. (2006). Heavy metal uptake by Scirpus Littoralis Schrad from fly ash dosed and metal spiked soils. Environmental Monitoring and Assessment, 121, 363–380.  Blaylock, M. J., Salt, D. E., Dushenkov, S., Zakharova, O., Gussman, C., Kapulnik,Y., Ensley, B. D. and Raskin, I. (1997). Enhanced accumulation of Pb in Indian mustard by soil– applied chelating agents. Environ. Sci. Technol., 31, 860–865.  Boonyapookana, B., Parkplan, P., Techapinyawat, S., DeLaune, R. D. and Jugsujinda, A. (2005). Phytoaccumulation of lead by sunflower (Helianthus annuus), tobacco (Nicotiana tabacum), and vetiver (Vetiveria zizanioides). J. Environ. Sci. Heal., 40, 117-137.  Chaney, R. L., Malik, M., Li, Y. M., Brown, S. L., Brewer, E. P. and Angle, J. S. (1997). Phytoremediation of soil metals. Curr. Opin. Biotech., 8, 279–283.  Characklis, G. W. and Wiesner, M. R. (1997). Particles, metals and water quality in runoff from large urban watershed. J. Environ. Eng., 123, 753–759.  Dushenkov, V., Kumar, P. B. A. N., Motto, H. and Raskin, I. (1995). Rhizofiltration: the use of plants to remove heavy metals from aqueous streams. Environ. Sci. Technol., 29, 1239- 1245.  Ebbs, S. D and. Kochian, L. V (1997). Toxicity of zinc and copper to Brassica species: implications for phytoremediation. J. Environ. Qual., 26, 776–781.  Ebbs, S. D., Lasat, M. M., Brandy, D. J., Cornish, J., Gordon, R., and Kochian, L. V. (1997). Heavy metals in the environment: Phytoextraction of cadmium and zinc from a contaminated soil. J. Environ. Qual., 26, 1424–1430.   83 Fakayode, S. O. and Olu-Owolabi, B. I. (2003). Heavy metal contamination of roadside topsoil in Osogbo, Nigeria, its relationship to traffic density and proximity to highways. Environmental Geology, 44, 150-157.  Hallberg, M., Renman, G.and Lundbom, T. (2007). Seasonal variations of ten metals in highway runoff and their partition between dissolved and particulate matter. Water Air Soil Pollut., 181, 183–191.  Hamlin, R. L. and Parker, A. V. (2006). Phytoextraction potential of Indian mustard at various levels of zinc exposure. Journal of Plant Nutrition, 29, 1257–1272.  Hoagland, D. R. and Arnon, D. I. (1950). The water-culture method of growing plants without soil, revised edition, Circular 347. Berkeley, CA: California Agricultural Experiment Station.  Hopkins, W. G. (2000). A scale of magnitudes for effective statistics. In: A New View of Statistics. Online publication. Internet Society for Sport Science. (http://www.sciencedirect.com/science?_ob=RedirectURL&_method=externObjLink&_lo cator=url&_plusSign=%2B&_targetURL=http%253A%252F%252Fwww.sportsci.org%25 2Fresource%252Fstats%252Feffectmag.html).  Huang, J. W. and Cunningham, S. D. (1996). Lead phytoextraction: species variation in lead uptake and translocation, New Phytol., 134, 75–84.  January, M. C., Cutright, T. J., Keulen, H. V. and Wei, R. (2008). Hydroponic phytoremediation of Cd, Cr, Ni, As, and Fe: Can Helianthus annuus hyperaccumulate multiple heavy metals? Chemosphere, 70 (3), 531-537.  Kabata-Pendias, A. and Pendias, H. (2001). Trace Elements in Soils and Plants. CRC Press, Boca Raton.  Kumar, P. B. A. N., Dushenkov, V., Motto, H. and Raskin, I. (1995). Phytoextraction: The use of plants to remove heavy metals from soils. Environ. Sci. Technol., 29, 232-238.  Legret, M and Pagotto, C. (1999). Evaluation of pollutant loadings in the runoff waters from a major rural highway. The Sci. Total Environ., 235, 143–150.  Levy, D. B., Redente, E. F. and Uphoff, G. D. (1999). Evaluating the phytotoxicity of Pb-Zn tailings to big bluestem (Andropogon gerardii Vitman) and switchgrass (Panicum virgatum L). Soil Science, 164, 363-375.  Lin, J., Jiang, W. and Liu, D. (2003). Accumulation of copper by roots, hypocotyls, cotyledons and leaves of sunflower (Helianthus annuus L). Bioresource Technology, 86, 151–155.  Liu, Y. G., Zhang, H. Z., Zeng, G. M., Huang, B. R. and Li, X. (2006). Heavy metal accumulation in plants on Mn mine tailings. Pedosphere, 16, 131-136.  Lintern, M. J., Butt, C. R. M. and Scott, K. M. (1997). Gold in vegetation and soil-three case studies from the goldfields of southern Western Australia. J. Geochem. Explor., 58, 1-14.  84 Luttmerding, H. A. (1980). Soils of the Langley-Vancouver map area. BC Soil Survey Report No. 15, pp. 20-21.  Macfie, S. M. and Welbourn, P. M. (2000). The cell wall as a barrier to uptake of metal ions in the unicellular green alga Chlamydomonas reinhardtii (Chlorophyceae). Arch. Environ. Contam. Toxicol., 39,. 413–419.  Marchiol, L., Assolari, S., Sacco, P. and Zerbi, G. (2004). Phytoextraction of heavy metals by canola (Brassica napus) and radish (Raphanus sativus) grown on multi contaminated soil. Environ Pollut., 132 (1), 21-27.  Marschner, B., Henke, U. and Wessolek, G. (1995). Effects of ameliorative additives on the adsorption and binding forms of heavy-metals in a contaminated topsoil from a former sewage farm. Z. Pflanz. Bodenkunde, 158, 9–14.  Mattina, M. J. I., Lannucci-Berger, W., Musante, C. and White, J. C. (2003). Concurrent plant uptake of heavy metals and persistent organic pollutants from soil. Environ. Pollut., 124, 375–378.  McIntyre, T. (2003). Phytoremediation of heavy metals from soils. In Advances in Biochemical engineering, Biotechnology, Phytoremediation, Scheper, T (eds), Springer-Verlag, New York, pp. 97–123.  Meyers, D. E. R., Auchterlonie, G. J., Webb, R. I. and Wood, B. (2008). Uptake and localisation of lead in the root system of Brassica juncea. Environ. Pollut., 153 (2): 323-332  Ministry of Environment, Lands and Parks. (1995). Criteria for Managing Contaminated Sites in British Columbia. Victoria, British Columbia, Queen’s Printer for B.C.  Preciado, H. F. and Li, L. Y. (2006). Evaluation of metal loading and bioavailability in air, water and soil along two highways of British Columbia, Canada. Water Air Soil Pollut., 172, 81– 108.  Richman, L. A. 1997. Niagara River Mussel Biomonitoring Program (1995). Ministry of Environment, Ontario, Queen’s Printer for Ontario.  Robert, W., Nunez, S., Blankinship, L. A. and. Gauthier, J. (2003). Stability of straw coated with sulfide and used for treatment of heavy metal-contaminated runoff, in conference Proceedings: Green Chemical Engineering Topical Conference, National AIChE Meeting, New Orleans, LA, pp. 139-151.  Salt, D. E., Blaylock, M., Kumar, P. B. A. N., Dushenkov, V., Ensley, B. D., Chet, I. and Raskin, I. (1995). Phytoremediation: a novel strategy for the removal of toxic metals from the environment using plants. Biotechnology, 13, 468–474.  Sansalone, J. J., and Buchberger, S. G. (1997). Partitioning and first flush of metals in urban roadway storm water. J. Environ. Eng., 123, 134–143.   85 Shu, W. S., Ye, Z. H., Lan, C. Y., Zhang, Z. Q. and Wong, M. H. (2002). Lead, zinc and copper accumulation and tolerance in populations of Paspalum distichum and Cynodon dactylon. Environ Pollut., 120, 445–453.  Smith, R. A. H. and Bradshaw, A. D. (1979). The use of metal tolerant plant populations for the reclamation of metalliferous wastes. Journal of Applied Ecology, 16, 595–612.  Spirochova, I. K., Pucocharova, J., Kafka, Z., Kubal, M., Soudek, P. and Vanek, T. (2003). Accumulation of heavy metals by in vitro cultures of plants. Water Air Soil Pollut., 3, 269- 276.  Stoltz, E. and Greger, M. (2002). Accumulation properties of As, Cd, Cu, Pb and Zn by four wetland plant species growing on submerged mine tailings. Environ Exp Bot., 47, 271– 280.  Tandy, S., Schulin, R. and Nowack, B. (2006). The influence of EDDS on the uptake of heavy metals in hydroponically grown sunflowers. Chemosphere, 62(9), 1454-1463.  Uren, N. C. and Reisenauer, H. M. (1998). The role of root exudates in nutrient acquisition. Adv. Plant Nutr., 3, 79–114.  Weng, G., Wu, L., Wang, Z., Luo, Y. and Christie, P. (2005). Copper uptake by four Elsholtzia ecotypes supplied with varying levels of copper in solution culture. Environment International, 31 (6), 880-884.  Wong, M. H. and Bradshaw, A .D. (1982). Comparison of the toxicity of heavy metals, using root elongation of rye grass, Lolium perenne. New Phytol., 91, 255-261.  Yoon, J., Cao, X., Zhou, Q. and Ma, L. Q. (2006). Accumulation of Pb, Cu, and Zn in native plants growing on a contaminated Florida site. The Sci. Total Environ., 368, 456 –464.  86 5. 4EXPLORATION OF PHYTOREMEDIATION AND ITS EFFECT ON MOBILITY OF METALS IN SOIL – A FRACTIONATION STUDY  5.1 Introduction  Pollution of the natural environment due to the release of metals from various sources is a global problem (Kabata-Pendias and Pendias, 2001). Sources of anthropogenic metal contamination include smelting of metalliferous ore, electroplating, gas exhaust, energy and fuel production, application of fertilizers and municipal sludge to land, industrial manufacturing and vehicular traffic (Singh et al., 2004; Sansalone et al., 1996). In urban areas, vehicular traffic is an important source of metal contamination in the environment (REC, 1998). High concentrations of metals, particularly lead, zinc, manganese, and copper in highway runoff result from the wear of brakes, tires and other vehicle parts, leakage of lubricants (Birch and Scollen, 2003; Sutherland et al., 2003) and exhaust emissions (Preciado and Li, 2006). Even though Mn is ubiquitous as a hydrous oxide, it was studied because of high Mn concentrations in roadside soils which can interfere with the normal life processes. Mn is from the methyl cyclopentadienyl manganese tricarbonyl (MMT), replacement of the tetraethyl lead (TEL) used as an anti-knock compound for gasoline engines in the early 1980s. Li et al. (2008) indicates that the concentration of Mn increased in the stream sediments and highway runoff. Metals are the most persistent constituents found in pavement runoff, and the transport of anthropogenic metal constituents by pavement runoff can adversely affect the quality of adjacent receiving waters and soils (Sansalone et al., 1996; Fakayode and Olu-Owolabi, 2003, Li et al., 2008). Exposure to high levels of these metals has been linked to adverse health effects on humans and wildlife (Bozkurt et al., 2000).  The environmental hazards of metal pollution in soil depend on the geochemical and biochemical properties of a given metal and are related to pedogenic and biogenic processes operating over time, which determine their mobility and bioavailability (Adriano et al., 2004; Kabata-Pendias and Pendias, 2001). Water-soluble and exchangeable forms of metals in soils are  4  A version of this chapter has been published. Padmavathiamma, P.K. and Li, L.Y. (2009) Exploration of phytoremediation and its effect on mobility of metals in soil – a fractionation study. Land Contamination and Reclamation, 17(2): 223-236.     87 considered readily mobile and available to plants, whereas metals incorporated into crystalline lattices of clays appear to be relatively inactive (Shuman, 1985). Other forms of metals, precipitated as carbonate, occluded in Fe, Mn, and Al oxides, or complexed with organic matter, could be strongly bound in soils, depending on the actual composition, physical and chemical properties of soil (Li and Thornton, 2001). The remediation of metal-contamination in soils along highways extending to thousands of kilometers is challenging. Existing metal remediation techniques such as solidification/stabilization, flotation, soil washing, electroremediation, bioleaching and microbial surfactants (Singh and Cameotra, 2004; Zoubeir et al., 2007) are expensive and not feasible for extended distances and large contaminated areas. Phytoremediation is a relatively sustainable and eco-friendly method not only to reduce the hazard associated with the presence of excess metals, but also to improve the soil quality and restore its functionality (Salt et al., 1995; Ebbs and Kochian, 1998; Singh et al., 2004). Using phytostabilisation to reduce the environmental impact by holding the metal-pollutants at the source location in non-mobile forms by the growth of plants (Smith and Bradshaw, 1979) could be a feasible management method for soils along highways, because it requires minimal maintenance.  Most phytoremediation studies have focused only on the plant-uptake of metals at only one stage of plant growth. Even though it is known that plant roots exude organic compounds which mobilize metals in soils and have indirect effects on microbial activity, in turn, affect the soil properties (Uren and Reisenauer, 1988), there has been limited study on the effects of phytoremediation on metal fractions in soil. Only a few studies have focused on the redistribution of metals in various soil fractions due to plant growth, for example, Guptha and Sinha (2006) on Sesamum indicum, King et al. (2008) on the mobilisation of Zn and Pb by the growth of Salix, Populus and Alnus and Degryse et al. (2008) on the mobilisation of Zn and Cu by growing spinach (Spinacia oleracea L.) and tomato (Lycopersicon esculentum L.). There is no published study to the authors’ knowledge which examines the mobility or immobility of metals in soils at different stages of plant growth during phytoremediation. The quantity of metal associated with a particular soil fraction varies depending upon plant species, stage of plant growth, conditions of the site and plant uptake characteristics (King et al., 2008). These factors control the potential mobility of metals to water sources and food chain, causing health and environmental hazards (Adriano et al., 2004; King, 2008). Therefore, the present study is  88 focused on assessing the effect of growth of five different plant species on the redistribution of soil metal fractions for Cu, Pb, Mn and Zn at two growth stages in roadside soil. The study involved a systematic and comprehensive effort with the research protocol: (1) investigating the physico-chemical properties of the soil before and after plant growth, (2) assessing the distribution of metal fractions in the rhizosphere at two different growth stages under variable multi- metal contamination levels, (3) examining the relationship of soil-metal fractionation to physico-chemical properties of soil (4) assessing the plant-metal accumulation at different growth stages; and (5) examining the relationship of plant metal concentrations with soil-metal fractionation. This study provides information on the effect of plant growth on metal dynamics and the distribution of metal fractions in soil at two stages of plant growth. The results identify suitable plants for highway soil metals remediation.  5.2 Materials and methods  The studied soil was collected from the grassed and vacant yard of Surrey Fire Hall No. 5, located 1 km north of the intersection of TCH (Trans Canada Highway) with the 176 Street overpass in Surrey, British Columbia. This site has the same soil as the nearby highway intersection (Luttmerding, 1980). Soils with two different metal concentrations were studied: (a) B0, the original soil containing concentrations of Cu 52 mg/kg, Pb 93 mg/kg, Mn 215 mg/kg, Zn 70 mg/kg. (b) BA, the original soil spiked with addition of all four metals to give total Cu, Pb, Mn, and Zn concentrations of 80, 146, 408 and 148 mg/kg, respectively. The metal concentrations studied were based on the results from the preliminary studies of the highway site (Padmavathiamma et al., 2007), previous work on roadside soils (Fakayode and Olu-Owolabi, 2003; Preciado and Li, 2006) and the British Columbia Standards for B.C contaminated sites (B.C Ministry of Environment, 1995). Metal fractionation studies were conducted in soils before and after plant growth in pot experiments. The five plant species tested are common in temperate maritime climate, typically found along the west coasts at the middle latitudes of the world's continents and are also listed in the data base "Phytorem" (Environment Canada, 2003). They are Lolium perenne L (perennial rye grass), Festuca rubra L (creeping red fescue), Helianthus annuus L (sunflower), Poa pratensis L (Kentucky bluegrass) and Brassica napus L (rape). They were grown in B0 and BA soils separately. The detailed experimental program of the study is summarized in Table 5.1. Details of soil spiking, experiment layout and sampling are given in  89 chapter 4, section 4.2.1. At each sampling stage, i.e. at 90 and 120 DAS, destructive sampling was applied. After removing the plants, both shoot and root, the soil in the pot was taken out, mixed well and a representative sample was taken. Since the roots covered almost the full soil, the whole soil was considered as the rhizosphere soil, and there is no bulk soil. The soil samples were taken to the laboratory in an ice bucket and immediately refrigerated at 4°C. Metal fractionation in the soil was performed after estimating the moisture content of soil samples. The plant samples were thoroughly washed with running tap water and rinsed with de-ionized water to remove any soil/sediment particles attached to the plant surfaces. Shoots and roots were then separated and oven dried (at 70ºC) to constant weight. The dried tissues were weighed and ground for analysis of Cu, Pb, Mn and Zn.  Table 5.1 Experimental Program for identification of plant species for phytostabilisation Metals studied Metal concentrations Plant species Stages of sampling Chemical Analysis Cu  Pb  Mn  Zn B0  – Original soil with metal concentrations of 52, 93, 215 and 70 mg/kg of Cu, Pb, Mn and Zn respectively.  BA  – B0 soil spiked with metals to give total metal concentrations of 80, 146, 408 and 148 mg/kg of Cu, Pb, Mn and Zn respectively.   Lolium perenne L (perennial rye grass)  Festuca rubra L (creeping red fescue)  Helianthus annuus L (sunflower)  Poa pratensis L (Kentucky bluegrass)  Brassica napus L (rape)  90 DAS  120 DAS  Basic soil characteristics.  Metal fractionation by Selective Sequential Extraction.  Total soil metals.  Plant metal concentrations.  Plant uptake Experimental Design – Completely Randomized Design, 10 treatments (5 plant species and 2 metal concentrations) and 3 replications.  Soil and plant samples were collected at two stages of plant growth, 90 and 120 DAS days after sowing. Fallowed soils without plants, subjected to similar conditions like other treatments  90 were also kept for comparison and sampled at 90 and 120 DAS. The basic characteristics of the soil such as pH, electrical conductivity, texture, % Carbon, total N and CEC were measured based on standard procedures (Table 3.1). The total metals in the soil samples were estimated by the USEPA method (Smoley, 1992). Different metal fractions in soils: exchangeable, oxide, organic and residual fractions were estimated using selective sequential extraction, according to the procedure of Tessier et al. (1979), as modified by Preciado and Li (2006). Since the soil has a pH <6, the carbonate fraction was negligible. Hence, the carbonate extraction was combined with oxide extraction and reported as such. The plant samples were air dried and ashed as recommended by Lintern et al (1997). The ash was dissolved in 10 mL 1 M HCl and diluted to 50 mL with deionized water. Soil and plant extracts were analysed for Cu, Pb, Mn and Zn using a Varian Spectre AA 220 Multi-element Fast Sequential Atomic Absorption Spectrometer. Blanks, duplicate samples and reference materials were used to ensure the quality and accuracy of the chemical analysis of the plants and soils. The statistical significance of differences among means was determined by one- way analysis of variance (ANOVA) to compare the treatment effects on soil metal fractionation, total soil metal concentration as well as metal uptake by plants. Statistical significance was defined at the level of P ≤ 0.05. Correlation and regression analyses were conducted to establish the relationship between soil and plant metal concentrations. The strength of each relationship was interpreted according to the correlation classification of Hopkins (2000), namely negligible: 0.0–0.09; low: 0.1–0.29; moderate: 0.3–0.49; high: 0.5– 0.69; very high: 0.7–0.89; nearly perfect: 0.9–1.0. When R was statistically significant at P ≤ 0.05, an asterisk (*) is provided to denote the statistical significance.  5.3 Results and discussion  Basic characteristics of the soils studied are summarized in Chapter 4, Table 4.2. All plant species tested (Lolium, Festuca, Helianthus, Poa and Brassica) survived in B0, whereas only three (Lolium, Poa and Festuca) survived in BA. LB0 represents Lolium in B0 soil, LBA (Lolium in BA soil), FB0 (Festuca in B0 soil), FBA (Festuca in BA soil), PB0 (Poa in B0 soil), PBA (Poa in BA soil), HB0 (Helianthus in B0 soil) and BrB0 (Brassica in B0 soil). Hereafter these abbreviations are used in the text, figures and tables.   91  5.3.1 Physico-chemical properties of soil as influenced by plant growth  The pH and electrical conductivity of soils at 90 and 120 DAS are given in Figure 5.1. The initial pH values for B0 and BA soils were 5.3 and 5.0. After fallowing and maintaining the same moisture % as the treated plots, the pH was found to be 5.2 and 5.0 for B0 and 4.9 and 4.8 for BA soil at 90 and 120 DAS. With plant growth, soil pH ranged from 4.9-5.3 at 90 DAS and 5.3-5.7 at 120 DAS. Thus there was a decrease in soil pH at 90 DAS and an increase at 120 DAS from the initial value with plant growth, while in fallowed soils a decrease from the initial value was observed at both 90 and 120 DAS. The decrease in soil pH in fallowed soils and planted soil at 90 DAS may be due to re-wetting of air-dried soil leading to microbial stimulus and enhanced reactions such as nitrification. The increase in soil pH at 120 DAS in planted soils, when compared to fallowed soils, may be attributed to the effect of root exudates or microbial exudates in the rhizosphere, which are important from the phytostabilisation point of view. The decrease in electrical conductivity accompanied by plant growth may be due to the lowering of soluble nutrient and metal ions in the soil by plant absorption or partitioning in insoluble soil fractions. Variations in pH of soils grown with different plant species may be due to the differential uptake of cations and anions by the plants, coupled with release of H+ or HCO3- and OH- (Marschner, 1995). Soil pH is the “master variable” due to its potential to modify metal solubility / availability in many ways (McBride, 1994). It controls dissolution / precipitation reactions, regulates the ionisation of pH-dependent exchange sites on organic matter and oxide clay minerals and influences metal speciation in soils (Adriano et al., 2004; Conesa et al., 2006).   92 4.5 5.0 5.5 6.0 LB0 FB0 HB0 PB0 BrB0 LBA FBA PBA Treatment So il pH 90 DAS 120 DAS  0.0 0.5 1.0 1.5 2.0 LB0 FB0 HB0 PB0 BrB0 LBA FBA PBA Treatment El e c tr ic a l C o n du c tiv ity  (d S/ m ) 90 DAS 120 DAS Fallowed soil  Initial value     90 DAS   120 DAS Figure 5.1  (a) pH and (b) Electrical Conductivity of soils at 90 and 120 DAS. Error bars represent ±S.D of means of three replicates. F-values for pH and Electrical Conductivity are significant at P <0.05. Fallowed soils are soils without plants, but subjected to similar conditions like other treatments and sampled at 90 and 120 DAS. (b) (a)  93 5.3.2 Metal fractionation in Soils  The metals in the soils were categorized in four fractions: exchangeable, oxide and carbonate- bound, organic-bound and residual. The partitioning of Cu, Pb, Mn and Zn in fallowed soils (without plant growth) and in soils with plant growth at 90 and 120 DAS is presented in Figure 5.2. The exchangeable fraction was found to be higher in BA soil than in B0 soil for all four metals. The % distributions of metal fractions in fallowed soils are as follows: Cu mainly in organic and residual fractions, Pb in oxide and residual fractions, Mn in oxide fraction, Zn in exchangeable and oxide fractions. The changes in metal fractionation with plant growth are discussed below.  Cu – The results show that Cu was retained mainly in the organic fraction at 90 DAS (days after sowing) and oxide fraction at 120 DAS (Figure 5.2). Compared to B0 soil (4% exchangeable Cu), there was a significant reduction (P<0.05) of exchangeable Cu (<1%) at 90 and 120 DAS due to the growth of Festuca (FB0 and FBA). In soils growing Festuca (FB0 at 90 DAS, FB0 and FBA 120 DAS), more than 90 % of exchangeable Cu was re-distributed to oxide Cu (see Figure 5.2), indicating the suitability of Festuca as a phytostabilising plant for Cu. There was an increase of exchangeable Cu in soils growing Poa (7%) and Brassica (6%) at 120 DAS. This is not desirable because of the increased mobility and bioavailability of this fraction. The increase of exchangeable Cu in Poa and Brassica growing soils can be attributed to the effect of root exudates and extruded protons in decreasing the soil pH and increasing the solubility of Cu. The organic Cu in soils growing Lolium (LB0) and Poa (PB0) were 45 and 47% at 90 DAS, whereas it was only 27-32% in soils growing Festuca (FB0), Helianthus (HB0) and Brassica (BrB0). This may be due to the well developed fibrous root system of Lolium and Poa, contributing to high soil organic matter and hence to organic-bound Cu in the soil. This is consistent with the observations of several other authors (Balasoiu et al., 2001 and Clemente et al., 2006), indicating that Cu forms very stable complexes with organic matter. Cu associated with the organic fraction can be mobile or immobile depending on the organic fraction to which it is complexed. The acidic pH of the soil in the present study (4.7 – 5.2 at 90 DAS) tended to retain less Cu as stable organic complexes, re-distributing the organic fraction to the oxide fraction at 120 DAS. This may explain the high partitioning of Cu in the oxide fraction at 120 DAS, regardless of the plant species. The differential partitioning of Cu in various soil fractions during the growth of different plant species can be attributed to the differential release of root exudates. These root  94 secretions have the ability to mobilize metals by shifting the equilibrium between different forms (Kabata-Pendias and Pendias, 2001). The ways in which plant roots alter the local metal chemistry in the rhizosphere have an impact on changing the oxidation state of metals (Marschner, 1995).  Pb- Pb partitioning through sequential extractions showed that a minor fraction of metal was exchangeable and that complexation with oxides, together with organic matter, was high. Although there was an increase in the organic fraction of Pb due to plant growth, the oxide fraction dominated at 90 DAS, and increased further at 120 DAS by re-distribution from exchangeable and organic forms (Figure 5.2). This may be due to the effect of plants in enhancing Pb immobilisation through stimulation of microbial activity, uptake into roots, redox reactions and formation and precipitation of insoluble Pb compounds in the rhizosphere (Wenzel et al., 1999). Poa soils (PB0 and PBA) had the least exchangeable Pb (1%) at both 90 and 120 DAS. The highest exchangeable Pb was observed in soils growing Helianthus (HB0) and Brassica (BrB0) at 120 DAS (Figure 5.2). As in the case of Cu, the highest oxide Pb was observed in Festuca-growing soils (FB0 and FBA) at 120 DAS and organic Pb in soils growing Lolium (LBA) and Poa (PBA) at 90 DAS. The re-distribution of more than 90 % of soluble or mobile (exchangeable) Pb to insoluble or immobile (oxide) Pb by the growth of Poa (PB0 and PBA at 120 DAS) reveals the suitability of Poa as a phytostabilising plant for Pb. Pb is similar in behaviour to Cu with respect to its tight bonding with the relatively insoluble fractions of the soil. However, Pb does not form stable organic complexes and hence the relative distribution in organic fraction was lower than that of Cu (Li et al., 2007). This is consistent with McBride and Martınez (1994) who reported that the main mechanisms responsible for lead immobilisation are chemisorption on oxides and silicate clays and precipitation as carbonates, hydroxides or phosphates.  Mn - The dominance of Mn in the oxide fraction with very little partitioning in the organic fraction occurred at 90 and 120 DAS for each of the plant species. Poa (PB0 and PBA) tended to retain more Mn in the exchangeable and oxide fractions of the soil, compared to the other plant species. As the growth stage advanced from 90 to 120 DAS, the exchangeable Mn increased further in soils growing Poa (PB0 and PBA) and Festuca (FB0 and FBA), whereas it decreased significantly (P<0.05) in Lolium-growing soils (LB0 and LBA, Figure 5.2). The low content of exchangeable Mn in Lolium-growing soils is notable, since the mobility of metal fractions is in  95 the order, exchangeable > carbonate specifically adsorbed > Fe-Mn oxide > organic sulfide > residual (Li and Thornton, 2001). Thus Lolium appears to be a suitable plant for phyto- stabilising Mn. The oxide fraction of Mn dominated in Festuca-growing soils (FB0 and FBA) at both 90 and 120 DAS. At 120 DAS, the organic fraction of Mn was further reduced by re- distribution to the oxide and exchangeable fractions (Figure 5.2). This may due to the fact that Mn in soil is largely associated with fulvic acid, and the Mn2+ bound to these compounds is highly ionized (Cheshire et al., 1977).  Zn - The predominance of Zn in the exchangeable and oxide fraction is evident from Figure 5.2. As the growth stage advanced from 90 to 120 DAS, the proportion of the oxide and organic fractions increased as a result of re-distribution from exchangeable and residual fractions. The exchangeable fraction of Zn was relatively high at 90 DAS, with a decline at 120 DAS (Figure 5.2). In Festuca growing soils (FBA), the exchangeable fraction decreased from 22% at 90 DAS to 14 % at 120 DAS. A significant reduction (P<0.05) in exchangeable Zn was found in soils growing Poa (PB0 and PBA) both at 90 and 120 DAS, giving evidence of the suitability of Poa for phytostabilisation of Zn. Unlike other metals which partitioned predominantly among oxides, organic and residual phases, Zn seems to be more in the exchangeable form (Alvarez et al., 2002; Su and Wong, 2003). The acidic pH, together with secretion of root exudates, might explain the high proportion of Zn associated with the exchangeable fraction. Since the pH was acidic for the soils examined (4.7-5.2 at 90 DAS and 5.3-5.7 at 120 DAS), there is greater risk of Zn mobilization compared to the other metals studied.   96    Exch. Oxide Organic Residual  Figure 5.2 Metal fractionation (%) in soil by the influence of plant growth at 90 and 120 DAS. n = 3, F-values are significant at P <0.05. LB0 (Lolium B0 soil), LBA (Lolium BA soil), FB0 (Festuca B0 soil), FBA (Festuca BA soil), PB0 (Poa B0 soil), PBA (Poa BA soil), HB0 (Helianthus B0 soil) and BrB0 (Brassica B0 soil).     0% 20% 40% 60% 80% 100% B0 BA Zn  fra ct io n at io n  (% ) 0% 20% 40% 60% 80% 100% LB0 LBA FB0 FBA HB0 PB0 PBA BrB0 Zn   fr a c tio n a tio n  (% ) 0% 20% 40% 60% 80% 100% LB0 LBA FB0 FBA HB0 PB0 PBA BrB0 Zn   fr a c tio n a tio n  (% ) 0% 20% 40% 60% 80% 100% LB0 LBA FB0 FBA HB0 PB0 PBA BrB0 M n   fr a c tio n a tio n  (% ) 0% 20% 40% 60% 80% 100% B0 BA Pb  fra ct io n at io n  (% ) 0% 20% 40% 60% 80% 100% LB0 LBA FB0 FBA HB0 PB0 PBA BrB0 Pb  fr a c tio n a tio n  (% ) 0% 20% 40% 60% 80% 100% LB0 LBA FB0 FBA HB0 PB0 PBA BrB0 Pb  fr a c tio n a tio n  (% ) 0% 20% 40% 60% 80% 100% LB0 LBA FB0 FBA HB0 PB0 PBA BrB0 M n   fr a c tio n a tio n  (% ) 0% 20% 40% 60% 80% 100% B0 BA M n  fra ct io n at io n  (% ) 0% 20% 40% 60% 80% 100% LB0 LBA FB0 FBA HB0 PB0 PBA BrB0 Cu   fr a c tio n a tio n  (% ) 0% 20% 40% 60% 80% 100% LB0 LBA FB0 FBA HB0 PB0 PBA BrB0 Cu   fr a c tio n a tio n  (% ) 0% 20% 40% 60% 80% 100% B0 BA Cu  fra ct io n at io n  (% ) 90 DAS 120 DAS Fallowed soil  97 5.3.3 Metal comparison  The metal fractionation studies summarized above indicate a significant partitioning of metals to insoluble forms by the growth of Festuca for Cu, Poa for Pb and Zn and Lolium for Mn. There was a decrease in the exchangeable fraction and an increase in the oxide and organic fraction of metals in soils as a result of plant growth. However the increase was more in the organic fraction for Cu and Pb and in the oxide fraction for Mn and Zn. This indicates that Cu and Pb are better able to form stable organic complexes than Mn and Zn for the range of conditions investigated. Thus the dynamic metal fractions were exchangeable, oxide and organic, while the residual fraction was not significantly affected by plant growth. Since the aim of the present study was to explore phytoremediation based on mobility/bioavailability of soil metals at different stages of plant growth, the total metal concentration values of soil obtained after harvesting plants are less significant, and the decrease observed often lies within the range of experimental error (Calace et al., 2002). However, a comparison of final metal concentrations in soils grown with the five plant species shows that maximum reduction was achieved by the growth of Lolium for Cu and Pb, Poa for Mn and Lolium and Brassica for Zn. The ability of Poa pratensis to phytoremediate Mn-contaminated soil was reported previously by Liu et al. (2006), while the effect of Brassica napus in reducing soil Zn was reported by Ebbs et al. (1997) and Ebbs and Kochian (1998). If the total metal concentration in soil is taken as a criterion to judge the impact of metal contamination, it implies that all forms of metals have an equal impact on the environment. Soluble, exchangeable and chelated species of metals are the most mobile components in soil, facilitating their migration and availability to the environment (Li and Thornton, 2001). Hence the efficiency of plants in influencing mobile/immobile partitioning of metals is important in soil remediation, in addition to lowering the total soil metal concentrations.  5.3.4 Relationship between soil pH and metal fractions  pH is the most important factor controlling the distribution of metals among different forms, and it has the largest influence on the bio-availability of heavy metals as a result of its strong influence on solubility and speciation of metals in the soil. The relationships between soil pH and various metal fractions at 120 DAS are given in Figure 5.3. Soil pHs for five treatments – LB0, FB0, HB0, PB0 and BrB0 – were correlated with the soil metal fractions (exchangeable, oxide, organic and residual). The pH was the same (5.4) for FB0 and PB0 at 120 DAS as shown in Figure 5.3. The relationship of soil pH to metal fractions shows the differential behaviour of  98 metals to soil pH. It is seen that the exchangeable (mobile) fraction of Cu, Pb, Mn and Zn gave negative correlations with the pH of the soil, significant (P<0.05) in the case of Cu and Zn (R = - 0.620 and -0.741). This reveals that mobile metal fractions (exchangeable) decrease with increase in soil pH. Each unit increase in pH results in approximately a two-fold decrease in the soluble concentration of Cu and Zn (Sanders et al. 1986). Oxide fractions of Pb and Zn gave significant positive correlations (P<0.05) with soil pH (R = 0.762 and 0.792).                 Exch Oxide Organic Residual   Residual Organic Oxide Exchangeable 0.172 -0.014 -0.340 -0.620* Cu 0.076 0.594* 0.762* -0.441 Pb 0.136 0.667* -0.289 -0.539 Mn -0.230 -0.657* 0.792* -0.741* Zn *Correlation coefficient was statistically significant at P 0.05. Figure 5.3. Effect of soil pH on soil metal fractions (exchangeable, oxide, organic and residual) at 120 DAS. (a) soil pH and Cu fractions, (b) soil pH and Pb fractions, (c) soil pH and Mn fractions, (d) soil pH and Zn fractions. Correlations between organic fractions of Pb and Mn and soil pH are also positive and significant (R = 0.594 and 0.667). Thus the relative partitioning of metals to the more immobile 0 5 10 15 20 25 5.0 5.2 5.4 5.6 5.8 C u   fra c tio n s  (m g/ kg ) 0 10 20 30 40 50 5.0 5.2 5.4 5.6 5.8 Pb   fra c tio n s  (m g/ kg ) 0 20 40 60 80 100 120 5.0 5.2 5.4 5.6 5.8 Soil pH M n   fra c tio n s (m g/ kg ) 0 5 10 15 20 25 5.0 5.2 5.4 5.6 5.8 Soil pH Zn   fra c tio n s  (m g/ kg ) (a) (b) (c) (d)  99 forms in Festuca-growing soils for Cu, Poa-growing soils for Pb and Zn and Lolium-growing soils for Mn, with extension of plant growth from 90 to 120 DAS can be explained by the increase in soil pH over this period (Figure 5.2). An increase in exchangeable Mn, with increase in soil pH from 5.2 to 5.7 can be seen in Figure 5.3. This may be because Mn can occur in more than one valence state and the oxidized state precipitates by the formation of hydroxides (or hydrous oxides). Up to pH 6.0, the hydroxides will not precipitate and the solubility of Mn increases (Brady and Weil, 1996).  5.3.5 Relationship of plant metal concentrations to soil metal fractions  Chapter 4 examined the metal concentrations (mg/kg dry weight) and metal uptake (µg/pot) of all five studied plants. The present study further explores the relationship between metal concentrations in plants and their fractions in soil. Although correlations were found between all studied metal fractions and plant metal concentrations (both root and shoot), only the most significant correlations are presented in Figure 5.4. This figure shows how the soil metal fractionation contributes to plant absorption and, thereby, exposure to the surrounding environment. Differential behaviour of metals in influencing root and shoot absorption is evident from Figure 5.4. In the case of Cu, oxide and organic Cu contribute to plant absorption, whereas for the other three metals (Pb, Mn and Zn), exchangeable and oxide fractions contribute to plant absorption. The strengths of the correlations reveal the extent of contribution of each soil metal fraction to plant metal concentrations. Significant positive correlations (P < 0.05) were observed between oxide Cu and root Cu (R = 0.774), organic Cu and root Cu (R = 0. 619), exchangeable Pb and root Pb (R = 0. 490), oxide Pb and root Pb (R = 0.652), exchangeable Mn and root Mn (R = 0.807), oxide Mn and shoot Mn (R = 0.898), exchangeable Zn and root Zn (R = 0.822) and oxide Zn and shoot Zn (R = 0.941). These relationships indicate the mobility or bioavailability of the respective metal fractions in the soil. The results suggest that the exchangeable and oxide- bound metals are of greater environmental importance than the organic and residual forms, because of their positive and significant correlations with plant metal concentrations. However, organic Cu has a significant relationship with plant Cu concentration, indicating the instability of organic Cu complexes formed at the acidic pH of the soil utilized in this study.       100 Figure 5.4 Relationship between plant metal concentrations (in root and shoot) and soil metal fractions. (a1) root Cu and oxide Cu; (a2) root Cu and organic Cu; (b1) root Pb and exchangeable Pb; (b2) root Pb and oxide Pb; (c1) root Mn and exchangeable Mn; (c2) shoot Mn and oxide Mn; (d1) root Zn and exchangeable Zn; (d2) shoot Zn and oxide Zn.                             101 30 40 50 60 70 80 90 100 110 5 10 15 20 25 30 Organic  Cu (mg/kg) R o o t C u  (m g/ kg ) 30 40 50 60 70 80 90 1 2 3 4 5 6 7 Exchangeable Pb (mg/kg) R o o t P b (m g/ kg ) 200 400 600 800 1000 5 10 15 20 25 30 Exchangeable Mn (mg/kg) R o o t M n  (m g/ kg ) 0 200 400 600 800 0 5 10 15 20 25 Exchangeable Zn (mg/kg) R o o t Z n  (m g/ kg )     40 50 60 70 80 90 100 110 5 10 15 20 25 30 Oxide Cu (mg/kg) R o o t C u  (m g/ kg ) 30 40 50 60 70 80 90 25 35 45 55 Oxide Pb (mg/kg) R o o t P b (m g/ kg ) 100 300 500 700 900 1100 75 100 125 150 175 Oxide Mn (mg/kg) Sh o o t M n  (m g/ kg ) 0 200 400 600 800 10 20 30 40 50 Oxide Zn (mg/kg) Sh o o t Z n  (m g/ kg ) (a2) (b1) (b2) (c1) (c2) (d1) (d2) (a1) B0 - 90 DAS B0 - 120 DAS BA - 90 DAS BA - 120 DAS  102 Relationship Regression Equation Correlation Coefficient (R) Oxide Cu vs Root Cu y=34.508Ln(x) – 27.186 0.774* Organic Cu vs Root Cu y=31.202Ln(x) – 19.608 0.620* Exchangeable Pb vs Root Pb y=12.53Ln(x) + 45.203 0.490 Oxide Pb vs Root Pb y=31.352Ln(x) – 52.021 0.652* Exchangeable Mn vs Root Mn y=375.33Ln(x) – 423 0.887* Oxide Mn vs Shoot Mn y=1072.2Ln(x) – 4710.5 0.898* Exchangeable Zn vs Root Zn y=200.35Ln(x) – 148.55 0.822* Oxide Zn vs Shoot Zn y=459.78Ln(x) – 1164.2 0.941* *Correlation coefficient was statistically significant at P 0.05  There are various reaction rates that control the partitioning of metals among various soil forms, such as exchangeable, oxide, organic and residual (Adriano et al., 2004). The quantity of metal associated with a particular fraction in the soil depends on the amount of metals added, the duration of the addition, soil pH, % organic matter, clay content and soil moisture content. Plant growth plays a major role in orchestrating these factors directly by their chemical and physiological effects and indirectly by the rhizobacteria associated with them (Marschner, 1995). Because, the distribution and association of metals with various soil fractions have a direct effect on mobility and bioavailability, continuous monitoring of metal partitioning during different plant growth stages is essential for successful phytoremediation.  Significant partitioning of metal fractions to insoluble forms was achieved by the growth of Festuca for Cu, Poa for Pb and Zn and Lolium for Mn. Hence Lolium, Poa and Festuca can be identified as potential phytostabilisation agents for metal-contaminants (Cu, Pb, Mn and Zn).  103 5.4 Conclusions and recommendations  • Soil pH increased from 90 to 120 days after sowing while electrical conductivity decreased over the same period.  • There was a decrease in the exchangeable fraction and an increase in the oxide and organic fractions of metals in soils as a result of plant growth. Oxide fraction of metals dominated in Festuca growing soils, organic fraction in soils growing Lolium and Poa and exchangeable fraction in soil growing Helianthus and Brassica.  • Exchangeable fractions of Cu, Pb, Mn and Zn were significantly negatively correlated with soil pH. Oxide fractions of Cu and Pb, and oxide as well as exchangeable fractions of Mn and Zn gave positive and significant correlations with plant metal concentrations, revealing the environmental importance of these metal fractions.  • A significant partitioning of metals to insoluble forms was observed by the growth of Festuca for Cu, Poa for Pb and Zn and Lolium for Mn. Metals partitioned more to insoluble forms with the prolongation of plant growth from 90 to 120 DAS.  • Lolium, Poa and Festuca appear to be suitable for phytostabilisation of Cu, Pb, Mn and Zn in moderately-contaminated acidic soils.  This study assists in understanding the effects of plant growth on metal dynamics (distribution of metal fractions) in soils with variable multi-metal contamination levels at two different stages of plant growth. Since the distribution and association of metals with various soil fractions directly affect mobility and bioavailability, continuous monitoring of metal partitioning during different plant growth stages is essential to prevent associated risks. Addition of metal-specific natural soil amendments that can modify the pedogenic processes and influence the plants to immobilise metals is recommended for future study.  104 5.5 References  Adriano, D. C., Wenzel, W. W., Vangronsveld, J. and Bolan, N. S. (2004). Role of assisted natural remediation in environmental cleanup. Geoderma, 122, 121–142.  Alvarez, E. A., Mochon, M. C., Sanchez, J. C. J. and Rodriguez, M. T. (2002). Heavy metal extractable forms in sludge from wastewater treatment plants. Chemosphere, 47, 765–775.  Balasoiu, C. F., Zagury, G. J. and. Deschenes, L. (2001). Partitioning and speciation of chromium, copper and arsenic in CCA-contaminated soils: influence of soil composition. Science of The Total Environment, 280, 239–255.  B.C Ministry of Environment, Lands and Parks. (1995). Criteria for Managing Contaminated sites in British Columbia – Contaminated Sites Remediation and assessment Section- Environmental Protection Department.  Birch, G. E. and Scollen, A. (2003). Heavy metals in road dust, gully pots an Parkland soils in a highly urbanised sub-catchment of Port Jackson, Australia. Australian Journal of Soil Research, 41, 1329-1342.  Bozkurt, S., Moreno, L. and. Neretnieks, I. (2000). Long term processes in waste deposits. Science of The Total Environment, 250, 101–121.  Brady, N. C. and Weil, R. R. (1996). The Nature and Properties of Soils. Prentice-Hall, Inc. New Jersey, pp. 495-498.  Calace, N., Petronio, B. M., Picciolo, M. and Pietroletti, M. (2002). Heavy metal uptake by barley growing in polluted soils: relationship with heavy metal speciation in soils. Commun. Soil Sci. Plant Anal., 33(1&2), 103–115.  Cheshire, M. V., Berrow, M. L., Goodman, B. and Mundie, C. M. (1977). Metal distribution and nature of some Cu, Mn and V complexes in humic and fulvic acid fractions of soil organic matter. Geochim. Cosmochim. Acta, 41, 1131.  Clemente, R., Almela, C.and Bernal, P. M. (2006). A remediation strategy based on active phytoremediation followed by natural attenuation in a soil contaminated by pyrite waste. Environmental Pollution, 143(3), 397-406.  Conesa, H. M., Faz, Á. and Arnaldos, R. (2006). Heavy metal accumulation and tolerance in plants from mine tailings of the semiarid Cartagena-La Unión mining district (SE Spain). Science of The Total Environment, 366, 1–11.  Degryse, F., Verma, V. K. and. Smolders, E. (2008). Mobilization of Cu and Zn by root exudates of dicotyledonous plants in resin-buffered solutions and in soil. Plant and Soil, 306, 69–84.  Ebbs, S. D. and Kochian, L. V. (1998). Toxicity of zinc and copper to Brassica species: Implications for Phytoremediation. J. Environ. Qual. 26, 776–781.   105 Ebbs, S. D., Lasat, M. M., Brady, D. J., Cornish, J., Gordon, R. and Kochian, L. V. (1997). Phytoextraction of cadmium and zinc from a contaminated soil. J. Environ. Qual., 26, 1424-1430.  Environment Canada (2003). Phytorem – Potential Green solutions for metal contaminated sites, green biotechnology, CD - rom.  Fakayode, S. O and Olu-Owolabi, B. I. (2003). Heavy metal contamination of roadside topsoil in Osogbo, Nigeria: its relationship to traffic density and proximity to highways. Environmental Geology, 44, 150-157.  Gupta, A. K. and Sinha, S. (2006) Chemical fractionation and heavy metal accumulation in the plant of Sesamum indicum (L.) var. T55 grown on soil amended with tannery sludge: Selection of single extractants. Chemosphere, 64 (1), 161-173.  Hoagland, D. R. and Arnon, D. I. (1950). The water culture method for growing plants without soil. Columbia agriculture Experiment Station Circular, 347, 1-32.  Hopkins, W. G. 2000. A scale of magnitudes for effective statistics. In: A New View of Statistics. Online publication. Internet Society for Sport Science. (http://www.sciencedirect.com/science?_ob=RedirectURL&_method=externObjLink&_lo cator=url&_plusSign=%2B&_targetURL=http%253A%252F%252Fwww.sportsci.org%25 2Fresource%252Fstats%252Feffectmag.html)  Kabata-Pendias, A. and Pentias, H. (2001). Trace elements is soils and plants. CRC Press, Boca Raton.  King, R. F., Royle, A., Putwain, P. D. and. Dickinson, N. M. (2008). Changing contaminant mobility in a dredged canal sediment during a three-year phytoremediation trial. Environmental Pollution, 143 (2), 318-326.  Li, X. D. and Thornton, I. (2001). Chemical partitioning of trace and major elements in soils contaminated by mining and smelting activities. Appl. Geochem., 16, 1693-1706.  Li, Q. S., Wu, Z. F., Chu, B., Zhang, N., Cai, S. S. and Fang, J. H. (2007). Heavy metals in coastal wetland sediments of the Pearl River Estuary, China. Environmental Pollution, 149, 158-164.  Li, L. Y., Hall, K., Yuan, Y., Mattu, G., McCallum, D. and Chen, M. (2008). Mobility and Bioavailability of Trace Metals in the Water-Sediment System of the Highly Urbanized Brunette Watershed. Water, Air, and Soil Pollution, 197, 249-266.  Lintern, M. J., Butt, C. R. M. and Scott, K. M. (1997). Gold in vegetation and soil-three case studies from the goldfields of southern Western Australia. J. Geochem. Explor., 58, 1.  Liu, Y. G., Zhang, H. Z., Zeng, G. M., Huang, B. R. and Li, X. (2006). Heavy metal accumulation in plants on Mn mine tailings. Pedosphere, 16, 131-136.   106 Luttmerding, H. A. (1980). Soils of the Langley-Vancouver map area. BC Soil Survey Report No. 15, pp. 20-21.  Marschner, H. (1995). Mineral Nutrition of Higher Plants. Academic Press, London.  McBride, M. B. (1994). Environmental Chemistry of Soils. Oxford University Press, New York.  McBride, M. B and Martınez, C. E. (1994). Copper phytotoxicity in a contaminated soil: remediation tests with adsorptive materials. Sci. Technol., 34, 4386–439.  Padmavathiamma, P. K., Li, L.Y. and Lavkulich, L. (2007). Heavy metal contamination and potential of local plants for phytoremediation along Highways. Biogeochemistry of Trace Elements: Environmental Protection, Remediation and Human Health, Edited: Y. Ahu, N. Lepp and R Naidu. 9th International Conference on the Biogeochemistry of Trace Elements (9th IOBTE), Beijing, China, pp.648-649.  Preciado, H. F. and Li, L. Y. (2006). Evaluation of metal loadings and bioavailability in air, water and soil along two Highways of British Columbia, Canada. Water, Air, and Soil Pollution, 172, 81–108.  REC (Regional Environment Centre) (1998) Rationale for the phase-out of lead in gasoline. Retrieved July 2, 2002, from World Wide Web: http://www.rec.org/ REC/Publications/LeadOut/chapter21.html.  Salt, D. E., Blaylock, M., Kumar, P. B. A. N.,  Dushenkov, V., Ensley, B. D.., Chet, I. and Raskin, I. (1995). Phytoremediation: a novel strategy for the removal of toxic metal from the environment using plants. Biotechnology, 13, 468–474.  Sanders, J. R., McGrath, S. P. and Adams, T. M. (1986). Zinc, Copper and Nickel concentration in rye grass grown on sludge- contaminated soils of different pH. Journal of the Science of Food and Agriculture, 37, 961-968.  Sansalone, J. J., Buchberger, S. G., and Al-Abed, S. R. (1996). Fractionation of heavy metals in pavement run off. The Sci. Total Environ., 189/190, 371–378.  Shuman, L. M. (1985). Fractionation method for soil microelements. Soil Sci., 140, 11-22.  Singh, P and Cameotra, S. S (2004). Enhancement of metal bioremediation by use of microbial surfactants. Biochemical and Biophysical Research Communications, 319, 291–297.  Singh, S., Sinha, S., Saxena, R., Pandey, K. and Bhatt, K. (2004). Translocation of metals and its effects in the tomato plants grown on various amendments of tannery wastes: evidence for involvement of antioxidants. Chemosphere, 57, 91–99.  Smith, R. A. H. and Bradshaw, A. D. (1979). The use of metal tolerant plant populations for the reclamation of metalliferous wastes. Journal of Applied Ecology, 16, 595–612.  Smoley, C. K. (1992). Methods for the determination of metals in environmental samples. U.S. Environmental Protection Agency. Cincinnati, Ohio.  107  Su, D. C. and Wong, J. W. C. (2003). Chemical speciation and phytoavailability of Zn, Cu, Ni and Cd in soil amended with fly-ash stabilized sewage sludge. Environ. Int., 29, 895–900.  Sutherland, R. A., Day, J. P. and Bussen, J. O. (2003). Lead concentrations, isotope ratios and source apportionment in road deposited sediments, Honolulu, Oahu, Hawaii. Water, Air, and Soil Pollution, 142, 165-186.  Tessier, A., Cambell, P. G. C. and Bisson, M. (1979). Sequential extraction procedures for the speciation of particulate trace metals. Anal. Chim., 51, 844–851.  Uren, N. C. and Reisenauer, H. M. (1998). The role of root exudates in nutrient acquisition. Adv. Plant Nutr., 3, 79–114.  Wenzel, W. W., Lombi, E. and Adriano, D. C. (1999). Biogeochemical processes in the rhizosphere: role in phytoremediation of metal-polluted sites. In: Heavy metal stress in plants – From molecules to ecosystems. M.N.V. Prasad and J. Hagemeyer (eds) Springer- Verlag, Heidelberg, Berlin, New York, pp. 273-303.  Zoubeir, L., Adeline, S., Laurent, C. S., Yoann, C., Truc, H. T., Benoît, L. G. and Federico, A. (2007). The use of the Novosol Process for the treatment of polluted marine sediment. Journal of Hazardous Materials, 148 (3), 606-612.  108 6. 5EFFECT OF AMENDMENTS ON PHYTOAVAILABILITY AND FRACTIONATION OF COPPER AND ZINC IN CONTAMINATED SOIL  6.1 Introduction  Copper (Cu) and zinc (Zn) are essential elements for human health, as components of many metalloenzymes and respiratory pigments. They play an important role in mammalian cellular metabolism (Buch et al., 2008). Their homeostasis in plant cells at excess levels has been reported by Palmer and Guerinot (2009). High concentrations of these metals originate mainly from mining (Prasad and Freitas, 2003). Other anthropogenic sources include industries, agriculture (via fertilizers and pesticides), incineration of wastes, vehicular traffic, as well as dressings of sewage sludge and pig slurries (Ahluwalia and Goyal, 2007; Varrica et al., 2003; Vargoa et al., 2005). They can cause long-term risks to ecosystems and threaten human health. In human beings, excess Cu causes epigastric pain, gastrointestinal effects and hemolytic anaemia (Turnlund, 1999), whereas excess Zn causes tachycardia, vascular shock, dyspeptic nausea, pancreatictis and damage of hepatic parenchyma (Barone et al., 1998; Salgueiro et al., 2000). In plants, excess copper causes toxic effects by catalyzing the production of highly toxic hydroxyl radicals from intracellularly generated hydrogen peroxide and affecting membrane properties (Prasad et al., 2001), whereas excess Zn results in stunting of roots, lignification of epidermal cells and increased permeability of root membranes (Pahlsson, 1989).  Many technologies have been developed to treat and remediate Cu- and Zn-contaminated soils (Cao et al., 2003; Peng et al., 2009). Phytoremediation is a cost-effective, environmentally- friendly technology, which could maintain the biological and functional integrity of soil after remediation (Pilon-Smits, 2005; Padmavathiamma and Li, 2007). Reducing the mobility and phyto-availability of contaminants (e.g. trace elements), through adsorption, absorption and accumulation in roots and precipitation in the rhizosphere, by growing metal-tolerant plants is termed phytostabilisation (Smith and Bradshaw, 1979). This technology is often associated with soil amendment treatments for in situ stabilisation of contaminants - “aided phytostabilisation”  5  A version of this chapter has been published. Padmavathiamma, P.K. and Li, L.Y. (2009) Phytostabilisation – a sustainable remediation for zinc toxicity in soils. Water Air Soil Pollution, 9(3-4), 253-260.   109 (Bes and Mench, 2008). Some amendments such as compost, cyclonic ash, zerovalent iron grit, and phosphate, as well as plants such as Festuca rubra and Agrostis capillaris have been reported to be effective in stabilising metal-contaminants (Smith and Bradshaw, 1979; Mench et al., 2006; Brown et al., 2004; Adriano et al., 2004; Simon, 2005; Kumpiene et al., 2007; Hartley and Lepp, 2008; Bes and Mench, 2008). Few studies have been conducted on the phytostabilisation of metal-contaminated sites, metal bioavailability and metal bioaccessibility to animals (Mench et al. 2006; Brown et al. 2004). However limited comprehensive studies, using plants and natural agricultural amendments to identify an integrated, sustainable, eco-friendly and cost-effective package for phytostabilisation, have been performed in acid soils of south- west British Columbia. The present study was undertaken in a highway soil contaminated with Cu, Pb, Mn and Zn using natural agricultural amendments such as compost, lime and phosphate individually and in combination, together with three previously-identified phytoremediating plant species, Lolium perenne L (perennial rye grass), Festuca rubra L (creeping red fescue) and Poa pratensis L (Kentucky blue grass) (Padmavathiamma and Li, 2009a). The soils were spiked with multi- metals to simulate an actual contaminated site. This chapter focuses on Cu and Zn only; whereas the next chapter considers Pb and Mn. The objectives were to: (1) investigate the effect of amendment addition on Cu and Zn accumulation in plants, and (2) assess the effect of soil-amendment–plant interaction on the mobility and phyto-availability of these metals in soil. The aging effect of the spiked soil on metal-contamination is not undertaken in the present study. pH is the most important parameter determining the effect of aging on Zn partitioning in soils, and in soils with a low pH, aging has little effect on Zn bioavailability (Lock and Janssen, 2003). The main research tasks were to (1) estimate the metal concentrations and metal uptake by plants, (2) assess the translocation properties and accumulation characteristics of these metals in plants with and without amendment, (3) investigate the distribution of metal fractions in the soil with and without plant growth and amendment addition, and (4) examine the relationship between -  soil metal fractions and plant metal concentrations, biometric characteristics and plant metal concentrations, soil pH and soil metal fractions, and total soil metal concentration and plant Enrichment Coefficient (EC).   110 6.2 Materials and methods  A bulk soil sample of 0-15 cm top layer was collected from the backyard of Surrey Fire hall No. 5, near a major highway intersection (HW 1 with 176 street in Surrey, British Columbia). This soil, referred to hereafter as B0, was found to be contaminated with Cu (52 mg/kg), Pb (93 mg/kg), Mn (215 mg/kg) and Zn (70 mg/kg). This soil was spiked with Cu, Pb, Mn and Zn at 30, 50, 200 and 80 mg/kg, respectively to conform to the A-level British Columbia standards for contaminated sites (Ministry of Environment, B.C, 1995), resulting in total Cu, Pb, Mn, and Zn concentrations of 80, 146, 408 and 148 mg/kg, respectively (designated BA). Amendments such as lime, phosphate and compost were added to the BA soils individually and in combination. Dolomite (finely ground) was the liming material and the source of P was CaHPO4.2H2O (41% P2O5). The compost used was City of Vancouver Yard Trimming Compost (pH - 6.4; electrical conductivity - 3.2 dSm-1; C/N ratio - 21.3, Cu – 1.2 mg/kg, Zn - 42 mg/kg, Fe – 61 mg/kg and Mn - 146 mg/kg). The detailed experimental program is summarized in Table 6.1. The nomenclature used for various treatments/conditions is given in Table 6.1. The abbreviations used for different treatments in the text, tables and figures are B0 (original soil), BA (spiked soil), BAL (spiked soil + lime), BAP (spiked soil + phosphate), BAO (spiked soil + compost) and BALPO (spiked soil + lime + phosphate + compost). The details of amendment treatments are given in “Application of amendments”, Appendix C.  Table 6.1 Experimental Program for soil-plant-amendment interaction Metals studied Conditions/Treatments Plant species Stage of sampling • Cu  • Pb  • Mn  • Zn • Original soil with multi-metal concentrations (52 mg/kg Cu, 93 mg/kg Pb, 215 mg/kg Mn and 70 mg/kg Zn) – B0 • Original soil spiked with multi-metals to give total concentrations (80 mg/kg Cu, 146 mg/kg Pb, 408 mg/kg Mn and 148 mg/kg Zn) - BA. • BA plus lime (10 tons/ha) - BAL • BA plus phosphate (135 kg P2O5/ha) - BAP • BA plus compost (10 tons/ha) - BAO • BA plus lime plus phosphate plus compost (combined application) - BALPO) • Poa pratensis  • Lolium perenne  • Festuca rubra   • 90 days after sowing  Design – Completely Randomized Design. 18 treatments (6 conditions and 3 plant species) with 3 replications.  111 The plant species investigated were Lolium perenne L (perennial rye grass), Festuca rubra L (creeping red fescue) and Poa pratensis L (Kentucky blue grass). Fifty-four plastic pots of 1 kg size were used for the experiments. The weight of each pot without soil was determined before filling it with 1 kg soil. The soil in each pot was thoroughly mixed with the required quantity of amendments and the moisture content brought to the field moisture capacity (36%). Each pot was then kept for two days for equilibration and seeds sown (0.5 g/pot).  An experiment with 18 treatments and three replications was conducted in a greenhouse from August 2006 to November 2006, in a completely randomized design. The summary of weather conditions during this period is given in Appendix C, Table 2. Soil and plant samples were collected at 90 DAS (days after sowing). Shoots and roots were then separated and oven dried (70°C) to constant weight. The dried tissues were weighed and ground for metals analysis. Various biometric characters such as length of root, number of branches per root, root weight, length of shoot, shoot weight and root/shoot ratios were recorded.  The original soil (B0) was analysed for basic physico-chemical characteristics such as pH (water), electrical conductivity, % organic matter, available phosphorus and total metal contents (Table 3.1). The procedure of Tessier et al (1979), as modified by Preciado and Li (2006), was adopted for selective sequential extraction. Different metal fractions were: exchangeable, carbonates and oxide, organic, and residual. The plant samples were air dried and ashed by the method outlined by Lintern et al., 1997. The ash was dissolved in 10 mL 1 M HCl and diluted to 50 mL with de-ionized water. Soil and plant extracts were analysed for Cu, Pb, Mn and Zn concentrations by means of a Varian Spectre AA 220 Multi-element Fast Sequential Atomic Absorption Spectrometer. Quality checking and control were performed using blanks, duplicate samples and standard solutions.  The statistical significance of differences among means was determined by one-way analysis of variance (ANOVA) to compare the treatment effects on soil metal speciation, plant metal concentration, as well as metal uptake by plants. In order to assess the phytostabilisation efficiency, the Enrichment Coefficient (EC) of root (Croots/Csoil,, the ratio of root concentration to soil concentration) and shoot (Cshoots/Csoil, ratio of shoot concentration to soil concentration) and  112 Translocation Factor (TF = Cshoots/Croots, ratio of shoot concentration to root concentration) were calculated (Kumar et al., 1995). ECroot   = [Metal]root/[Metal]soil  ECshoot = [Metal]shoot/[Metal]soil  TF       = [Metal]shoot/[Metal]root   6.3 Results and discussion The texture of the soil was sandy clay loam and its classification was Luvisolic Humoferric Podzol according to the Canadian System of Soil Classification (1998). Basic characteristics were: pH – 5.6, electrical conductivity – 0.61 dS/m, % carbon – 1.5 and available phosphorus extracted by Bray 1 – 10.4 mg/kg. Total metal concentrations in the original soil (B0) and spiked soil (BA) were: Cu – 52 and 80 mg/kg, Pb – 93 and 146mg/kg, Mn – 215 and 408 mg/kg and Zn – 70 and 148 mg/ kg, respectively. 6.3.1 Effect of soil amendments on metal concentrations and uptake in plants.  The effects of various treatments on Cu and Zn concentrations in plants are portrayed in Figure 6.1.    113  Figure 6.1 Metal concentrations in plants (in root and shoot). (a) Cu concentrations in Lolium. (b) Zn concentrations in Lolium, (c) Cu concentrations in Festuca, (d) Zn concentrations in Festuca, (e) Cu concentrations in Poa, (f) Zn concentrations in Poa. BA – spiked soil, BAL - spiked soil plus lime, BAP - spiked soil plus phosphate, BAO - spiked soil plus compost, BALPO - spiked soil plus lime, phosphate and compost. F significant at P<0.05 for both Cu and Zn.  Application of lime (BAL) decreased the Cu and Zn concentration in the root and shoot of all the three plant species. The decrease was significant (P<0.05) in Festuca for Cu and in Poa for Zn. The increase in soil pH (Figure 6.2) by the application of lime reduced the exchangeable or phyto-available fractions of Cu and Zn (discussed under 6.3.3), and reflected in the low plant d c b c b cd ab a d bc 0 40 80 120 160 BA BAL BAP BAO BALPO Treatments Cu  c o n c e n tr a tio n  (m g/ kg ) Root Shoot e cdcd a dcd c d b 0 40 80 120 160 BA BAL BAP BAO BALPO Treatments Cu  c o n c e n tr a tio n  (m g/ kg ) Root Shoot d c ab c b cd ab a cbc 0 40 80 120 160 BA BAL BAP BAO BALPO Treatments Cu  c o n c e n tr a tio n  (m g/ kg ) Root Shoot e a dc a fg ab d fg ab 0 200 400 600 800 BA BAL BAP BAO BALPO Treatments Zn  c o n c e n tr a tio n  (m g/ kg ) Root Shoot e a c d b fg bc fg a 0 200 400 600 800 BA BAL BAP BAO BALPO Treatments Zn  c o n c e n tr a tio n  (m g/ kg ) Root Shoot f ab de c g bab f ab 0 200 400 600 800 BA BAL BAP BAO BALPO Treatments Zn  c o n c e n tr a tio n  (m g/ kg ) Root Shoot (a) (b)  Lolium (c) (d)  Festuca (e) (f) Poa  114 tissue concentrations of Cu and Zn. The effectiveness of lime in increasing soil pH and reducing metal availability was previously reported by Kabata-Pendias and Pendias (1992).  5.0 5.5 6.0 6.5 7.0 7.5 8.0 B0 BA BAL BAP BAO BALPO Treatments So il pH Lolium Festuca Poa   Figure 6.2 Soil pH as influenced by plant growth and amendment application.* - F significant at P<0.05. B0 – Initial soil, BA – Spiked soil, BAL - Spiked soil plus lime, BAP - Spiked soil plus   phosphate, BAO - Spiked soil plus compost, BALPO - Spiked soil plus lime, phosphate and compost.  Festuca showed the highest Cu concentration, but the amendments significantly lowered (P<0.05) the values, with the lowest values achieved with combined application of amendments. The shoot Cu concentration, which ranged from 47 to 54 mg/kg in BA soil, decreased to <30 mg/kg as a result of combined application of amendments (BALPO). Concentrations of Cu greater than 40 mg kg−1 of dry matter may induce toxicity in plants, and may also cause toxic effects in animals (eg. sheep) feeding on them (Annenkov, 1982). Compared to BA, application of phosphate (BAP) increased plant Cu in Lolium and Poa, but decreased plant Zn in all the three plant species. This may be due to the fact that although phosphate application augmented the exchangeable Zn fraction of the soil, it may have had an antagonistic effect on Zn absorption by the plants. Similar findings, where amendment addition increased the exchangeable and soluble metal fractions in soils with no significant influence on plant concentrations, were reported by Vangronsveld et al. (2000) and Simon (2005). Compost application (BAO) reduced the plant concentration of Cu, providing evidence for Cu binding with insoluble organic  115 fractions (Hsu and Lo, 2000), whereas augmentation was found for plant Zn (Figure 6.1). Combined application of amendments (BALPO) significantly decreased the concentrations of Cu and Zn in plants, with Festuca exhibiting the lowest values for Cu and Poa for Zn (Figure 6.1). With the combined application of amendments, the plant metal concentration decreased by more than 40 % for Cu and 70 % for Zn, compared to BA soil.  The highest plant biomass (both root and shoot) for all three plant species was recorded with the combined application of the amendments (2.3 g for Lolium, 1.8 g for Festuca and 2.0 g for Poa). This increase in biomass may be because; favourable plant growth under ideal soil pH (Figure 6.2), created by the combined addition of amendments provides a conducive rhizospheric climate by sequestering excess metals, along with the fertilizing effect of lime and compost. Root and Shoot biomass, dry weight (DW) (g/pot) is given in Appendix C, Table 3.  The Cu and Zn uptake by plants are given in Figure 6.3. Even though the plant biomass was higher in treatments that received amendments in combination, the uptake of Cu and Zn by these plants was less than for plants that received only one amendment (Figure 6.3). The low metal uptake may be due to the low mobile or phyto-available metal fractions in the soil because of precipitation as hydroxides, phosphates or sequestration by organic ligands by combined amendment addition (BALPO). There was a significant increase in Cu and Zn uptake (P<0.05), when phosphate and compost were applied individually (Figure 6.3). This may be due to the favourable growth conditions, i.e., nutrient supply and soil physical properties, created by the application of these amendments.     116 bababcccc c. a a b c c ab ab c aa bcbcbc aa b b bc aa bb 0 200 400 600 800 1000 1200 BA BA L BA P BA O BA LP O BA BA L BA P BA O BA LP O BA BA L BA P BA O BA LP O Lolium Festuca Poa m e ta l u pt a ke  (m ic ro  gr a m s /p o t) Cu Zn  Figure 6.3  Metal uptake by plants. BA – Spiked soil, BAL - Spiked soil plus lime, BAP - Spiked soil plus phosphate, BAO - Spiked soil plus compost, BALPO - Spiked soil plus lime, phosphate and compost. Mean values, n = 3. Bars followed by different letters show significantly different statistical values (P<0.05) for means.  6.3.2 Accumulation of Cu and Zn in plants as characterized by EC and TF  The Enrichment Coefficient (EC) and Translocation Factor (TF) of the three plant species after different treatments are given in Table 6.2, and the relationship between total soil metal content and EC (ECroot and ECshoot) in plants is given in Figure 6.4. Means followed by different letters show significantly different statistical values (P<0.05). For eg. ECroot of BALPO in Lolium growing soil is significantly lower than BA, BAP and BAO while it is on par with BAL. Compared to BA, application of lime (BAL) significantly lowered the ECroot, ECshoot and TF values of Cu and Zn (P<0.05). On the other hand, compared to BA, application of phosphates (BAP) increased the EC values (both root and shoot) of Cu in all the three plant species, not significant though. Compost application (BAO) had an augmenting effect only on ECroot of Zn (5.90) in Poa (Table 6.2). The lowest ECroot, ECshoot and TF for Cu (0.70, 0.36 and 0.45, respectively) were by Festuca, and for Zn (0.97, 0.63 and 0.44, respectively) by Poa, in both cases with combined application of amendments (BALPO). Low EC and TF values reveal a low plant absorption as well as translocation of metals in the plants. The differences between plant species in metal accumulation and translocation may be due to different release of root exudates, e.g. organic acids, CO2 and H+ that can change soil pH and control the release or sequestration of metals (Kelly et al., 1998). The ionic competition, high soil pH and binding of metals by root exudates may be the reasons for the lower translocation of metals to the above ground parts of  117 plants in amendment-applied treatments (Kumpiene et al., 2007). Also indirect application of Ca through lime may enhance the Ca/metal competition and a higher cellular Ca which may contribute to membrane integrity, lowering the oxidative stress, for maintaining the cell homeostasis (Prasad, 2004).  Table 6.2 Enrichment Coefficient (EC) and Translocation Factor (TF) in different treatments by plant growth and amendment additions. Enrichment Co-efficient Translocation Factor (TF) Cu Zn Plant species Treatments ECroot ECshoot ECroot ECshoot Cu Zn B0 1.50a 0.71c 2.44d 2.35b 0.48d 0.96a BA 1.19b 0.64cd 4.25b 3.55ab 0.54cd 0.84bc BAL 0.87cd 0.48e 2.15de 0.83c 0.55cd 0.39ef BAP 1.25b 0.69c 2.88cd 2.44b 0.54c 0.84bc BAO 0.95c 0.67cd 3.87bc 3.79ab 0.71a 0.97a    Lolium  BALPO 0.78d 0.48e 1.41ef 0.73c 0.62bc 0.76c  B0 1.39ab 0.94a 3.29c 3.11b 0.68ab 0.95a BA 1.41a 0.67cd 4.74ab 4.03a 0.48d 0.85b BAL 0.73d 0.45ef 2.58cd 0.84c 0.56c 0.33f BAP 1.47a 0.65cd 5.39a 3.76ab 0.47d 0.69cd BAO 1.21b 0.67cd 5.01a 3.99a 0.56c 0.60d    Festuca  BALPO 0.70d 0.36f 1.12ef 0.74c 0.45d 0.66c  B0 0.85cd 0.63cd 2.79cd 2.65b 0.74a 0.95a BA 1.22b 0.58d 4.87b 4.33a 0.48d 0.89ab BAL 0.72d 0.41ef 1.96e 1.09c 0.52cd 0.59d BAP 1.36ab 0.87ab 4.57ab 4.24a 0.64b 0.93a BAO 1.10bc 0.80bc 5.90a 2.63b 0.73a 0.45e BALPO 0.91c 0.63cd 0.97f 0.63c 0.69ab 0.44e    Poa F * * * * * * Mean values, n = 3. *F significant at P<0.05. Significantly different statistical values (P<0.05) according to the Least Significance Test in each column are followed by different letters. B0 – Initial soil, BA – Spiked soil, BAL - Spiked soil plus lime, BAP - Spiked soil plus phosphate, BAO - Spiked soil plus compost, BALPO - Spiked soil plus lime, phosphate and compost.  The relationship between total soil metal concentration and ECroot and ECshoot (Figue 6.4) reveals that as the total metal concentration in the soil increased, ECroot and ECshoot decreased for both Cu and Zn. ECroot and ECshoot for Cu and Zn were significantly correlated (P <0.05) with total  118 metal concentrations in the soil (R values being 0.882 and 0.807 for Cu, and 0.674 and 0.811 for Zn, respectively).     Figure 6.4 Relationship between soil metal concentrations and Enrichment Coefficients (EC) for root and shoot  Such an accumulation pattern, when the higher soil metal concentrations do not cause higher metal accumulation by plants, is favourable for phytostabilisation. This can be explained by the plant’s inherent ability to immobilise metals and decrease its absorption when the soil metal concentrations are high (Salisbury and Ross, 1992). The immobilization of metals in polluted y = -3.2766Ln(x) + 15.436 R = 0.882 y = -1.8445Ln(x) + 8.7094 R = 0.807 0 0.4 0.8 1.2 1.6 70 75 80 85 90 Total Cu in the soil (mg/kg) En ric hm en t C o ef fic ie n t EC root EC shoot y = -8.5426Ln(x) + 44.645 R = 0.674 y = -8.5378Ln(x) + 43.498 R = 0.811 0 2 4 6 8 85 95 105 115 125 135 145 Total Zn in the soil (mg/kg) En ric hm en t C o ef fic ie n t EC root EC shoot  119 soils by the growth of grasses has been reported by Bogatu et al. (2007). On the contrary, there are reports of increased metal uptake under low levels of soil metals, when plant roots increase metal bioavailability by extruding protons, phytosiderophores, and organic acids, acidifying the soil and mobilising metals (Marschener, 1998; Garbisu and Alkorta, 2001). Grasses are known to excrete phytosiderophores and LMWOA (low-molecular-weight organic acids), which are chelating agents, that bind metals into metal-chelate complexes, making them more available for plant uptake at low concentrations of soil metals (Ma and Nomoto, 1996).  6.3.3 Metal fractionation in the soil  Cu and Zn fractionation in the soil due to plant growth alone is presented Table 6.3, and by plant growth and amendment addition is given in Figure 6.5. It is seen that when the soil was spiked, there was an increase in the oxide fraction for Cu, whereas for Zn, the increase was mainly in the exchangeable fraction (Table 6.3). Growth of Lolium enhanced the exchangeable Cu fraction, whereas Festuca had a lowering effect (Table 6.3), in agreement with results from a previous study (Padmavathiamma and Li, 2009b). When compared to BA, lime application (BAL) significantly reduced exchangeable fractions of Cu and Zn (P<0.05), with this reduction being highest in Festuca-growing soils for Cu and Poa-growing soils for Zn (Figure 6.5). Similar observations on the effect of lime in reducing the mobile metal fractions were reported by Hooda and Alloway (1996). Lime amendments increase soil pH (McBride et al., 1997) and favour the formation of oxides, metal-carbonate precipitates, and other complexes that decrease metal solubility and bio-availability (Mench et al., 2006). Application of phosphates (BAP) significantly increased the exchangeable fraction in soils growing Poa and Lolium for Cu and Lolium and Festuca for Zn (Figure 6.5). A decrease in exchangeable Zn by phosphate application (BAP) when compared to BA, in Poa growing soils was noticed. This may be due to the immobilization of Zn as Zn phosphates, with low solubility and high resistance to soil acidification (Impellitteri, 2005). There was a significant increase in the organic fraction of Cu with the addition of compost (BAO), the highest value being recorded by soils growing Poa (Figure 6.5). The organic-bound Cu fraction was found to account for 50% of the total soil Cu in the Poa grown soil with combined amendments (BALPO). This may be due to a greater number of fine roots when plant growth is promoted by addition of compost. The number of branches/roots recorded in Poa (18) was significantly higher compared to that in Lolium (12) and Festuca (10.3) in BAO. This increase in organic-bound Cu fraction in Poa grown soil may  120 reflect Cu storage in fine roots and root hairs. However for Zn, the amount partitioned in the organic fraction was less, even in compost-applied treatments. Organic amendments can contribute to metal immobilisation through the formation of stable complexes with OH or COOH groups on the solid surfaces of the organic polymers (Chirenje and Ma, 1999). However, this may promote metal mobility if complexes formed with the amendments are more soluble than without them (Hsu and Lo, 2000).  Table 6.3. % partitioning of Cu and Zn in soils with and without plant growth.    % Cu fractions in soil % Zn fractions in soil Treatments /Conditions   Exch. Oxide Organic Residual Exch. Oxide Organic Residual B0 4.4 22.1 33.5 40 11.9 43.5 18.5 26.1 BA 4.9 25.8 31 38.3 21.8 39.1 11.3 27.7 Soil alone    B0 9.4 16.7 34.1 39.8 12.2 25.6 23.3 38.9 BA 6.1 30.1 24.7 39.1 22.8 30.6 15.7 30.8 Soil + Lolium       B0 2.3 50.7 20.3 26.7 10.4 40.9 18.2 30.8 BA 2.1 43.6 17.6 36.7 24.1 41.7 13.1 20.7 Soil + Festuca     B0 5.9 15.6 35.6 42.8 4.1 40.2 18.4 36.9 BA 4.7 24.1 40.5 31 8.5 45.1 13.9 32.9 Soil + Poa    The major partitioning of each metal in a soil is in bold. n=3. F significant at P<0.05. B0 – Initial soil, BA – Spiked soil.       121    Figure 6.5 Partitioning of Cu and Zn in soil by the effect of amendments and plants. BA – Spiked soil, BAL - Spiked soil plus lime, BAP - Spiked soil plus phosphate, BAO - Spiked soil plus compost, BALPO - Spiked soil plus lime, phosphate and compost. Mean values, n = 3. F significant at P<0.05.  Among the different treatments, maximum immobilisation of Cu and Zn in the soil was achieved by the combined application of amendments (BALPO) with Festuca and Poa, respectively. Combining amendments can be beneficial for maintaining a favourable soil pH (Jackson and Miller, 2000) and increasing Cu sorption on clay mineral surfaces through ternary Cu-mineral- humic acids complexes (Hizal and Apak, 2006). Also, with the combined application, the Ca content of lime can bind to phosphorus, making it less available to plants and thereby reducing its influence on increasing the mobile metal fraction (Schnoor, 1996). In the present study, since Exch Oxide Organic Residual 0% 20% 40% 60% 80% 100% B A L B A P B A O B A LP O B A L B A P B A O B A LP O B A L B A P B A O B A LP O B A L B A P B A O B A LP O Amendments alone Amendments + Lolium Amendments + Festuca Amendments + Poa %  Cu  fra c tio n a tio n 0% 20% 40% 60% 80% 100% BA L BA P BA O BA LP O BA L BA P BA O BA LP O BA L BA P BA O BA LP O BA L BA P BA O BA LP O Amendments alone Amendments + Lolium Amendments + Festuca Amendments + Poa %  Zn  fra ct io n at io n  122 lime and compost were added in conjunction with phosphate, the lowering of pH by phosphate was nullified by lime, creating a favourable environment for metal immobilisation in the soil.  6.3.4 Relationship between soil pH and metal fractions  Lime application (BAL) increased soil pH from 5.4 to from 7.2; whereas phosphate addition (BAP) decreased the pH in Festuca growing soils from 5.4 to 5.1. Compost application (BAO) had also an augmenting effect on soil pH. The relationship between soil pH and Cu fractions in soil indicate that soil pH had a significant influence on the exchangeable and organic Cu fractions (Figure 6.6). Exchangeable Cu was significantly negatively correlated with pH (Table 6.4) in soils growing all three plant species, the highest correlation being for Lolium (R = - 0.976). The organic fraction of Cu in Poa growing soils showed a significant positive correlation with soil pH (R = 0.663). As in the case of Cu, exchangeable Zn was also significantly and negatively correlated with pH, the greatest correlation coefficient being for soils growing Lolium (R = -0.802). Other Zn fractions in the soil (oxide, organic and residual) were positively and significantly correlated with pH (Table 6.4) in Poa growing soils (R values being 0.902, 0.565 and 0.656 respectively).           123  Figure 6.6 Relationship between soil pH and metal fractions. (a) soil pH and Cu fractions in Lolium soil, (b) soil pH and Zn fractions in Lolium soil, (c) soil pH and Cu fractions in Festuca soil, (d) soil pH and Zn fractions in Festuca soil, (e) soil pH and Cu fractions in Poa soil, (f) soil pH and Zn fractions in Poa soil.    0 10 20 30 40 5.0 5.5 6.0 6.5 7.0 7.5 pH of soil Cu   fr ac tio n s (m g/ kg ) 0 10 20 30 40 5.0 5.5 6.0 6.5 7.0 7.5 pH of soil Cu   fr ac tio n s (m g/ kg ) 0 20 40 60 80 100 5.0 5.5 6.0 6.5 7.0 7.5 pH of soil Zn   fra c tio n s  (m g/ kg ) 0 20 40 60 80 5.0 5.5 6.0 6.5 7.0 7.5 pH of soil Zn   fra c tio n s  (m g/ kg ) Exch. Oxide Organic Residual 0 10 20 30 40 5.0 5.5 6.0 6.5 7.0 7.5 pH of soil Cu   fr ac tio n s (m g/ kg ) 0 20 40 60 80 5.0 5.5 6.0 6.5 7.0 7.5 pH of soil Zn   fra c tio n s  (m g/ kg ) (a) (b)  (c) (d)  (e) (f)  (Poa)  (Festuca)  (Lolium)  124 Table 6.4 Correlation coefficients between soil pH and metal fractions  Cu Zn Lolium Festuca Poa Lolium Festuca Poa Exchangeable -0.976* -0.835* -0.609* -0.802* -0.789* -0.781* Oxide 0.135 0.465 0.349 0.878* 0.716* 0.902* Organic 0.424 0.447 0.663* 0.417 0.355 0.565* Residual 0.445 0.581* 0.301 0.600* 0.647* 0.656* *Correlation coefficient was statistically significant at P<0.05  Thus, stability of Cu and Zn in soil was strongly pH dependent – the mobility increasing with decreasing pH (Kabata-Pendias and Pendias, 1992). Also, a significant influence of pH was observed on the organic fraction of Cu and on the oxide and exchangeable fractions of Zn (Figure 6.6). Growth of plants alter the pH conditions of the soil by releasing of organic acids, generating CO2 from root respiration, exuding protons, and by the imbalance in cation or anion release caused by excess anion or cation uptake (Kelly et al., 1998). Because speciation varies with pH, the metal ion concentration exerting a given toxic effect can be expressed as a function of pH (Lofts et al., 2004). Predicting pCu2+ (soil-solution free copper activity) as a function of total soil copper and soil pH is a better exposure indicator (Sauve et al., 1997).  6.3.5 Relationship of plant metal concentration to soil metal fractions  Correlations between plant metal concentrations and soil metal fractions are given in Table 6.5. Both root and shoot Cu concentrations were positively correlated with exchangeable Cu (R of 0.661 and 0.589, respectively) and oxide Cu (R of 0.750 and 0.634, respectively) in soil (Table 6.5). Even though organic-bound Cu in soil was higher than other fractions, it did not influence the plant Cu concentration, revealing the non-availability of Cu-organic complexes to plants. In the case of Zn, a significant positive correlation was observed between exchangeable Zn fraction in the soil and both root and shoot Zn (Table 6.5). A lack of correlation between oxide-bound Zn in the soil and plant Zn concentration indicated the unavailability of that soil fraction to the plants, like Cu-organic complexes.    125 Table 6.5.  Correlations between plant (root and shoot) metal concentrations and soil metal fractions.  Cu root Cu shoot Soil metal fractions Regression equation R Regression equation R Cu exch. y = 8.98 x + 52.1 0.661* y = 5.16 x + 33.8 0.589* Cu oxide y = 2.62 x + 28.2 0.750* y = 1.43 x + 21.5 0.634* Cu organic y = 0.11 x + 73.7 0.038 y = 0.12 x + 44.9 0.061 Cu residual y = 0.52 x + 64.9 0.132 y = -0.71x + 63.3 -0.277  Zn root Zn shoot  Regression equation R Regression equation R Zn exch. y = 17.5 x + 180 0.890* y = 12.3 x + 118 0.986* Zn oxide y = -1.93 x + 52 -0.164 y = -3.73 x + 479 -0.448 Zn organic y = -2.3 x + 470 -0.083 y = -8.9 x + 468 -0.450 Zn residual y = 2.51 x + 327 0.115 y = -0.63 x + 316 -0.040 *Correlation coefficient (R) was statistically significant at P<0.05.  6.3.6 Plant metal concentrations and biometric characteristics  The correlation coefficients between biometric characteristics and plant concentrations (Cu and Zn) appear in Table 6.6. In Poa and Lolium, shoot length had a positive correlation with plant Cu (both root and shoot), whereas root length was negatively correlated with plant Cu and Zn. In Festuca, the number of root branches was significantly negatively correlated with plant Cu (root and shoot). In the case of plant Zn, none of the biometric characters was found to have a pronounced influence in Festuca, whereas in Poa, Zn root was positively and significantly correlated with shoot length (R = 0.506), and Zn shoot was significantly negatively correlated with root length. (R = -0.556). Thus, a negative relationship was shown by root parameters such as root length and number of root branches on metal concentration in plants. Exudates released by plant roots can bind with metals transforming them into organo-metallic complexes, which are neither mobile nor bio-available. These are important aspects of phytostabilisation, since the efficiency of rhizosphere metal precipitation depends on the exposure of plant roots to the contaminated zones.  126  Table 6.6.  Correlation coefficients between plant biometric characters and metal concentrations.  Plant species Metal concentration Shoot length Root length Number of branches/root Cu root 0.891* -0.889* -0.091 Cu shoot 0.776* -0.778* 0.005 Zn root 0.499 -0.659* 0.232 Lolium  Zn shoot 0.332 -0.686* -0.083 Cu root -0.338 -0.556* -0.766* Cu shoot -0.287 -0.399 -0.825* Zn root 0.036 -0.094 -0.353 Festuca  Zn shoot -0.423 -0.124 -0.216 Cu root 0.699* -0.254 0.362 Cu shoot 0.682* -0.2359 0.315 Zn root 0.506* 0.100 0.364 Poa          Zn shoot 0.208 -0.556* 0.232 *Correlation coefficient was statistically significant at P 0.05. Root parameters contribute to rhizosphere metal dynamics and ultimately the effectiveness of plants for soil metal de-contamination (Uren and Reisenauer, 1998; Marschner, 1998). The amount and composition of root exudates released into the rhizosphere are highly variable and dependent on plant species, stage of plant growth, physico-chemical environment and metal toxicity stress (Marschner, 1998).  6.4 Conclusions and recommendations  The results demonstrate the effectiveness of soil-plant-amendment interaction in suppressing the availability of Cu and Zn in a contaminated acid soil. Specific findings were:  • Changes in soil pH due to the application of lime had a significant effect on the exchangeable fractions of Cu and Zn, organic fractions of Cu, and oxide fractions of Zn in soil. Addition of lime to the soil lowered the plant Cu and Zn concentrations.   127 • Phosphate application increased the exchangeable Cu content in soil, increased plant Cu, and decreased plant Zn.  • Application of compost significantly increased the organic fraction of Cu, especially in soils grown with Poa, while, no pronounced effect on Zn fractionation was observed.  • With combined amendments, the concentration of Cu in shoots and roots decreased by more than 40 % in Festuca, and by ~70 % for Zn in Poa.  • Maximum metal immobilisation was achieved in the soil by the combined application of all amendments, in conjunction with growth of Festuca for Cu and Poa for Zn.  • `Lowest EC and TF values were observed in Festuca for Cu and Poa for Zn with combined application of amendments.  Since the soil contained multiple metal contaminations, the different properties of the contaminants restrict the choice of possible amendments in order to avoid large pH fluctuations and consequent mobilization of one or more of the metals. The presence of Cu can decrease the stabilization efficiency of Zn due to competition for sorption sites. Thus the treatment efficiency of multi-element contaminated sites can be improved only by applying a combination of amendments. In such cases, lowering of soil pH due to phosphate can be countered by adding lime. However, there is need to confirm these results under field conditions.  The metal speciation due to aging in the spiked soil, the functioning of root metal transporters and the change in microbial biomass and mycorhization are suggested as future studies.   128 6.5 References  Adriano, D. C., Wenzel, W. W., Vangronsveld, J. and Bolan, N. S. (2004). Role of assisted natural remediation in environmental cleanup. Geoderma, 122, 121-142.  Ahluwalia, S. S. and Goyal, D (2007) Microbial and plant derived biomass for removal of heavy metals from wastewater. Biores. Technol., 98, 2243-2257.  Annenkov, B. N. (1982). Mineral feeding of sheep. In: V.I. Georgievskii, B.N. Annenkov and V.I. Samokhin, Editors, Mineral nutrition of animals, Butterworths, London, pp. 321–354.  Barone, A., Ebesh, O., Harper, R. G. and Wapnir, R. A. (1998). Placental copper transport in rats: effects of elevated dietary zinc on fetal copper, iron and metallothionien. J Nutr., 128(6), 1037–1041.  Bes, C. and Mench, M. (2008). Remediation of copper-contaminated topsoils from a wood treatment facility using in situ stabilization. Environmental Pollution, 156 (3), 1128-1138.  Bogatu, C., Lixandru, B., Andres, L., Masu, S., Mosoarca, G., Negrea, A. and Ciopec, M. (2007). Heavy metals immobilization in soils by using of supported volcanic tuff and of biosolid. Chem. Bull. Univ., 52(66), 1-2.  Brown, S., Chaney, R., Hallfrisch, J., Ryan, J. A. and. Berti, W. R. (2004). In Situ Soil Treatments to Reduce the Phyto- and Bioavailability of Lead, Zinc, and Cadmium. J. Environ. Qual., 33, 522-531.  Buch, A., Hawkesworth, C. J., Ragnarsdottir, Ragnarsdottir, V. K. and Brown, D. R. (2008). Re- partitioning of Cu and Zn isotopes by modified protein expression. Geochem Trans., 9, 11.  Cao, X., Ma, L. Q., Chen, M., Singh, S. P. and Harris, W. J. (2003).  Phosphate induced metal immobilization in a contaminated site. Environ Pollut., 122, 19–28.  Chirenje, T. and Ma, L. Q. (1999). Effects of acidification on trace metal mobility in a papermill ash amended soil. J. Environ. Qual., 28(3), 760–766.  Garbisu, C. and Alkorta, I. (2001). Phytoextraction: a cost-effective plant-based technology for the removal of metals from the environment. Bioresource Technology, 77(3), 229-236.  Hartley, W. and Lepp, N. W. (2008). Remediation of arsenic contaminated soils by iron-oxide application, evaluated in terms of plant productivity, arsenic and phytotoxic metal uptake. Science of The Total Environment, 390(1), 35-44.   129 Hizal, J. and Apak, R. (2006). Modeling of copper(II) and lead(II) adsorption on kaolinite-based clay minerals individually and in the presence of humic acid. Journal of Colloid and Interface Science, 295 (1), 1-13.  Hooda, P. S. and Alloway, B. J. (1996). The effect of liming on heavy metal concentrations in wheat, carrots and spinach. J Agric Sci Cambridge, 127, 289–294.  Hsu, J. H. and. Lo, S. L. (2000). Characterization and extractability of copper, manganese, and zinc in swine manure composts. J. Environ. Qual., 29, 447–453.  Impellitteri, C. A. (2005). Effects of pH and phosphate on metal distribution with emphasis on As speciation and mobilization in soils from a lead smelting site. Science of The Total Environment, 345 (1-3), 175-190.  Jackson, B. P and Miller, W. P. (2000). Effectiveness of phosphate and hydroxide for desorption of arsenic and selenium species from iron oxides, Soil. Sci. Soc. Am. J., 64, 1616–1622.  Kabata-Pendias, A. and Pendias, H. (1992) Trace elements in soils and plants, CRC Press, Boca Raton, Florida.  Kelly, E. F., Chadwick, O. A. and Hiliniski, T. E. (1998). The effect of plants on mineral weathering. Biogeochem., 42, 21-53.  Kumar, P. B. A. N., Dushenkov, V., Motto, H. and Raskin, I. (1995). Phytoextraction: The use of plants to remove heavymetals from soils. Environ. Sci. Technol., 29(5), 1232-1 238.  Kumpiene, J., Lagerkvist, A.. and Maurice, C. (2007). Stabilization of Pb- and Cu-contaminated soil using coal fly ash and peat. Environmental Pollution, 145, 365-373.  Lintern, M. J., Butt, C. R. M. and Scott, K. M. (1997). Gold in vegetation and soil-three case studies from the goldfields of southern Western Australia. J. Geochem. Explor., 58, 1.  Lock, K. and Janssen, C. R. (2003). Influence of aging on zinc bioavailability in soils. EnvironmentalPollution, 126(3), 371-374.  Lofts, S., Spurgeon, D. J., Svendsen, C. and Tipping, E. (2004). Deriving soil critical limits for Cu, Zn, Cd, and Pb: a method based on free ion concentrations. Environmental Science and Technology, 38, 3623–3631.  Ma, J. F. and Nomoto, K. 1996. Effective regulation of iron acquisition in graminaceous plants. The role of mugineic acids as phytosiderophores. Physiologia Plantarum, 97, 609-617.  Marschner, H. (1998). Mineral Nutrition of Higher Plants. Academic Press, London.  McBride, M. B., Suave, S. and Hendershot, W. (1997).Solubility control of Cu, Zn, Cd, and Pb in contaminated soils, Europ. J. Soil Sci., 48, 337–346.   130 Mench, M., Vangronsveld, J., Lepp, N., Bleeker, P., Ruttens, A. and Geebelen, W. (2006). Phytostabilisation of metal-contaminated sites. In Phytoremediation of Metal- Contaminated Soils, Echevarria G., Morel J.L., Goncharova N.(Eds.), Nato Science Series: IV: Earth and Environmental Sciences 68: Springer, The Netherlands. pp. 109-190.  Ministry of Environment, Lands and Parks. (1995). Criteria for Managing Contaminated Sites in British Columbia. Victoria, British Columbia, Queen’s Printer for B.C.  Padmavathiamma, P. K. and Li, L. Y. (2007). Phytoremediation Technology: Hyper- accumulation metals in plants. Water, Air, and Soil Pollution, 184, 105-126.  Padmavathiamma, P. K. and Li, L. Y. (2009a). Phytoremediation of metal-contaminated soil in temperate humid regions of British Columbia, Canada. International Journal of Phytoremediation, 11(6), 575-590.  Padmavathiamma, P. K. and Li, L. Y. (2009b). Phytoremediation and its Effect on Mobility of Metals in Soil: a Fractionation Study. Land Contamination and Reclamation, 17(2), 223- 236.  Pahlsson, A. B. (1989). Toxicity of heavy metals (Zn, Cu, Cd, Pb) to vascular plants. Water, Air, and Soil Pollution, 47, 287-319.  Palmer, C. M. and Guerinot, M. L. (2009). Facing the challenges of Cu, Fe and Zn homeostasis in plants. Nature Chemical Biology, 5, 333-340.  Peng, J. F., Song, Y. H., Yuan, P., Cui, X. Y. and Qiu, G. L. (2009). The remediation of heavy metals contaminated sediment. Journal of hazardous materials, 161(2-3), 633-40.  Pilon-Smits, E. A. H. (2005). Phytoremediation. Annual Review of Plant Biology, 56, 15–39.  Prasad, M. N. V. and Freitas, H. M. O (2003) Metal hyperaccumalation in plants - Biodiversity prospecting for phytoremediation technology. Electr. J. Biotechnol., 6, 285-321.  Prasad, M. N. V. (2004). Heavy metal stress in plants. Springer-Verlag Publisher Inc., Heiderberg, pp. 182-201.  Prasad, M. N. V., Greger, M. and Landberg, T. (2001). Acacia nilotica L. bark removes toxic metals from solution: Corroboration from toxicity bioassay using Salix viminalis L. in hydroponic system. International Journal of Phytoremediation, 3 (3), 289-300.  Preciado, H. F. and Li, L. Y. (2006). Evaluation of metal loadings and bioavailability in air, water and soil along two Highways of British Columbia, Canada. Water, Air, and Soil Pollution, 172, 81–108.  Salisbury, F. B. and Ross, C. W. (1992). Plant Physiology. Wadsworth Publishing Company, Belmont, CA.   131 Salgueiro, M. J., Zubillaga, M., Lysionek, A., Sarabia, M. I., Caro, R., Paoli, T. D., Hager, A., Weill, R. and Boccio, J. (2000). Zinc as an essential micronutrient: a review. Nutr Res., 20(5), 737–755.  Sauve, E, S., Mcbride, M. B., Norvell, W.A. and Hendershot, W.H. (1997). Copper solubility and speciation of in situ contaminated soils: effects of copper level, pH and organic matter. Water, Air, and Soil Pollution, 100, 133–149.  Schnoor, J. L. 1996. Modeling trace metals. In: Environmental modelling – Fate and transport of pollutants in water, air, and soil. John Wiley & Sons, Inc. U.S. pp. 381-451  Simon L. (2005): Stabilization of metals in acidic mine spoil with amendments and red fescue (Festuca rubra L.) growth. Environ. Geochem. Health, 27, 289–300.  Smith, R. A. H. and Bradshaw, A. D. (1979). The use of metal tolerant plant populations for the reclamation of metalliferous wastes. Journal of Applied Ecology, 16, 595–612.  Tessier, A., Cambell, P. G. C. and Bisson, M. (1979). Sequential extraction procedure for the speciation of particulate trace metals, Anal. Chim., 51, 844–851.  Turnlund, J. R. (1999). Copper. In Modern nutrition in health and disease, 9th ed., eds. M. E. Shils,S. A. Olson, M. Shike, and A. C. Ross,. Baltimore, MD: Williams & Wilkins, pp. 870– 878.  Uren, N. C and Reisenauer, H. M. (1998). The role of root exudates in nutrient acquisition. In: Tinker E, Lauchli A (eds) Advances in plant nutrition, vol 3. Praeger, New York, pp. 798– 814.  Vangronsveld, J., Ruttens, A., Mench, M., Boisson, J., Lepp, N. W., Edwards, R., Penny, C. and van der Lelie, D. (2000). In situ inactivation and phytoremediation of metal- and metalloid- contaminated soils: field experiments. In: D.L. Wise, D.J. Trantolo, E.J. Cichon, H.I. Inyang and U. Stottermeister, Editors, Bioremediation of Contaminated Soils, Marcel Dekker, New York, Basel. pp. 859–884.  Vargova, M., Ondrasovicova, O., Sasakova, N., Ondrasovic, M., Culenova, K and Smirjakova, S. (2005). Heavy metals in sewage sludge and pig slurry solids and the health and environmental risk associated with their application to agricultural soil. Folia Veterinaria, 49, 28-30.  Varrica, D., Dongarra, G., Sabatino, G. and Monna, F. (2003). Inorganic Geochemistry of Roadway Dust from the Metropolitan Area of Palermo, Italy. Environmental Geology, 44, 222-230.  132 7. 6PHYTOAVAILABILITY AND FRACTIONATION OF LEAD AND MANGANESE IN CONTAMINATED SOIL FOLLOWING APPLICATION OF THREE AMENDMENTS  7.1 Introduction  Pb and Mn are the two metals that have been used extensively in transport vehicles during the past few decades. The use of tetraethyl lead (TEL) as an antiknock compound for gasoline engines in the early 1970s and the subsequent replacement by methyl cyclopentadienyl manganese tricarbonyl (MMT), have led to considerable exhaust emissions of Pb and Mn. Both are released to the environment by other anthropogenic activities too. For example, Pb contamination results from mining and smelting activities, use of Pb in paints as well as from the disposal of municipal sewage sludge and industrial wastes enriched in Pb (Joint FAO/WHO Expert Committee on Food Additives, 2000; Ma et al., 1995). Because of the high binding strength of Pb to soil fractions, Pb is highly immobile in soil and it becomes virtually permanent, with a soil retention time of 150 to 5000 years (Friedland, 1990). Hence, even though the use of leaded gasoline was suspended several decades ago in North America and most of the industrialized world, many roadside soils remain contaminated with Pb (Sezgin et al., 2003). In Canada, the anthropogenic emissions of Mn amounted to 1225 tons, with approximately 75% from industrial facilities and 20% from gasoline-powered motor vehicles using MMT (Environment Canada, 1987). Of particular concern is the effect of transportation systems on the release of these metals to the surrounding environment.  Contamination of soil by lead is of major concern due to its high toxicity to humans and animals and its bioavailability through ingestion or inhalation. Relatively low concentrations of Pb in the blood can affect children's mental development, an effect that persists into adulthood (Needleman et al., 1990; Laidlaw et al., 2005). Exposure to high concentrations of Mn can lead to numerous health problems, including neurodegenerative disorders similar to Parkinson's disease (such as manganism) (US EPA, 2003). Phytoremediation is a cost-effective, environmentally friendly and ecologically sound remediation method (Baker et al., 1994) for  6  A version of this chapter has been accepted for publication. Padmavathiamma, P.K. and Li, L.Y. (2010). Phytoavailability and fractionation of lead and manganese in contaminated soil following application of three amendments. Bioresource Technology.   133 metal contaminated sites. Depending upon the conditions of the site, level of clean up required and plant species, the remediation method can be either containment or removal (Padmavathiamma and Li, 2007). Containment by in-situ immobilisation or in-place inactivation of contaminants using plants and amendments is phytostabilisation (Smith and Bradshaw, 1979; Arienzo et al., 2003). This may be suited for busy contaminated sites such as highway soils, where contaminant removal is not feasible and practical due to physical and financial constraints.  Many amendments and plants have been reported to be effective for the stabilisation of different metal-contaminants (Simon, 2005; Kumpiene et al., 2007). But a holistic approach involving suitable plants and natural amendments that can remediate the metal-contaminated sites and retain the functional and ecological integrity of soil is still lacking. Hence, the present study was undertaken in contaminated acidic soils of coastal British Columbia using natural agricultural amendments such as lime, phosphate and compost individually and in combination along with the plant species: Lolium perenne L (perennial rye grass), Festuca rubra L (creeping red fescue) and Poa pratensis L (Kentucky blue grass). These plants were identified to be suitable for phytostabilisation from a previous study (Padmavathiamma and Li, 2009). The objectives were: (1) to assess the effect of soil-amendment–plant interaction on the partitioning of Pb and Mn into various soil fractions and to evaluate the mobility and phyto-availability of these metals in soil, and (2) to assess the effect of soil improvement or amelioration on the accumulation characteristics and translocation properties of these metals in the plants. The studies generated sufficient data to suggest a cost effective package that can not only reduce the hazard associated with the presence of excess Pb and Mn, but also improve the soil quality and sustain its functionality.  7.2 Materials and methods  The present study was performed in soil collected from the backyard of Surrey Fire hall No. 5, near the main intersection of highway 1 with 176th Street in Surrey, British Columbia. This represents a busy site with respect to traffic counts (>80,000 vehicles/day), and it is highly contaminated with Cu, Pb, Mn and Zn (Preciado and Li, 2006). This chapter focuses on Pb and Mn, whereas the previous chapter considered Cu and Zn. The metal interaction studies by  134 Padmavathiamma and Li (2009) revealed a high degree of correlation between Cu and Zn, and limited correlation between Pb and Mn. Gasoline combustion may be the main source of Pb and Mn in highway soils. The original soil (B0) containing 52 mg/kg Cu, 93 mg/kg Pb, 215 mg/kg Mn, 70 mg/kg Zn, was spiked with further Cu, Pb, Mn and Zn, resulting in total measured Cu, Pb, Mn, and Zn concentrations of 80, 146, 408 and 148 mg/kg, respectively (designated BA), approximately matching the British Columbia Ministry of Environment (1995) A-level limits for contaminated sites. Details on the soil spiking with multi-metals and addition of soil amendments are given in Chapter 6. The plant species used for the study were Lolium perenne L (perennial rye grass), Festuca rubra L (creeping red fescue) and Poa pratensis L (Kentucky blue grass). The study was conducted as a pot experiment in a completely randomized design with 18 treatments and three replications (Table 6.1). The experiment was done in the greenhouse during the period, August 2006 to November 2006. Soil and plant samples were collected at 90 DAS (days after sowing).  Basic characteristics of the soil such as pH, electrical conductivity, organic carbon, available P and texture were estimated. The procedure of Tessier et al (1979), as modified by Preciado and Li (2006), was adopted for selective sequential extraction. The different metal fractions estimated were: exchangeable, carbonates and oxides, organic and residual. The plant samples were air dried and ashed according to method outlined by Lintern et al., 1997. The ash was dissolved in 10 mL 1 M HCl and diluted to 50 mL with de-ionized water. Soil and plant extracts were analysed for Pb and Mn using a Varian Spectre AA 220 Multi-element Fast Sequential Atomic Absorption Spectrometer.  The statistical significance of differences among means was determined by one-way analysis of variance (ANOVA) followed by least significant difference (LSD) tests. Correlation and regression analyses were conducted to establish the relationship between different parameters. When R was statistically significant at P ≤ 0.05, an asterisk (*) is provided to denote the statistical significance. In order to assess the efficiency of plants for phytostabilisation, the Enrichment Coefficient (EC) of root (Croots/Csoil,, the ratio of root concentration to soil concentration) and shoot (Cshoots/Csoil, ratio of shoot concentration to the soil concentration) and Translocation Factor (TF = Cshoots/Croots, ratio of shoot concentration to the root concentration) were calculated (Kumar et al., 1995).  135  7.3 Results and discussion  The chemical characteristics of the studied soils are given in Table 7.1.  Table 7.1. Physico chemical properties of the studied soils  Total metal concentrations (mg kg-1) Soil pH Electrical Conductivity (dSm-1) % Carbon Total N (%) Available P (mg kg-1) Cu Pb Mn Zn Original soil (B0) 5.6 0.61 1.50 0.15 10.4 52 93 215 70 Spiked soil (BA) 5.4 1.27 1.28 0.11 16.7 80 146 408 148  7.3.1 Metal concentrations and uptake in plants  The plant concentration (mg/kg) and plant uptake (µg/pot) of Pb and Mn (both root and shoot) are given in Table 7.2. Means followed by different letters indicated as superscripts in Table 7.2 show significantly different statistical values (P<0.05). The lowest plant concentrations, as well as uptake (both root and shoot) (significant at P<0.05), were observed in Lolium for Mn, when compared to Festuca and Poa. In the case of Pb, for B0 and BA soils, the lowest root concentration was observed in Poa (32 and 61 mg/kg, respectively), though not statistically significant. The combined application of amendments (BALPO) lowered the plant concentrations of Pb and Mn more than the individual additions of amendments. Lime amendments (BAL) reduced both plant Pb and Mn (concentration and uptake), whereas phosphate amendments (BAP) decreased the plant Pb and increased the plant Mn (concentration and uptake).        136  Table 7.2. Metal concentrations and metal uptake by the plants (root and shoot).  Mn Pb Plant concentration (mg/kg) Plant uptake (µg/pot) Plant concentration (mg/kg) Plant uptake (µg/pot) Plant species Conditions/ Treatments Root Shoot Root Shoot Root Shoot Root Shoot B0 276g 179j 146i 174h 36bc 17ab 19c 13bc BA 914cd 835e 475fg 592f 65a 18a 31a 12bc BAL 787d 639fg 535f 686ef 40b 8d 27a 8cd BAP 1887a 1170c 1460a 1474b 35bc 11c 25b 13bc BAO 974c 1003d 749d 1404b 44b 16ab 33a 22a     Lolium BALPO 535e 506h 267h 759e 27cd 9cd 20c 11c B0 297fg 246ij 151i 132h 41b 21a 20c 13bc BA 1051c 1023d 294h 429g 67a 20a 18c 8cd BAL 929cd 780ef 455g 678ef 45b 9cd 22bc 7d BAP 1973a 1283b 1210b 1308c 34bc 10cd 20c 10c BAO 1053c 1090cd 651e 1253c 40b 17a 22bc 19a    Festuca BALPO 861d 607g 542f 758e 30c 8d 19c 21a B0 390f 297i 205h 175h 32bc 19a 20c 11c BA 1691b 1766a 845cd 918d 61a 20a 32a 10c BAL 840d 686fg 529f 434g 42b 7d 26b 4d BAP 1985a 1209b 1207b 1414b 35bc 13b 22bc 15b BAO 1143c 1178bc 910c 1763a 49b 15b 38a 19a BALPO 861d 607g 542f 758e 30c 8d 19c 21a     Poa F * * * * * * * *  Mean values, n = 3. * F significant at P<0.05. Statistically significantly different values (P<0.05) according to the Least Significance Test. in each column are followed by different letters. B0 – Initial soil, BA – Spiked soil, BAL - Spiked soil plus lime, BAP - Spiked soil plus phosphate, BAO - Spiked soil plus compost, BALPO - Spiked soil plus lime, phosphate and compost.  The reduction in plant Pb by phosphate application may be due to the precipitation of Pb as Pb phosphate in the soil. The acidic pH of the studied soil (5.4) and the source of P, which is Ca HPO4. 2H2O (41 % P2O5) can contribute to the precipitation of Pb phosphate in the studied soil. Apart from the addition of phosphates to the soil, root exudates from plants contain phosphatase  137 enzymes that can convert organic P to phosphate in the rhizosphere (Haeussling and Marschner, 1989) and this free phosphate would be available to metal compounds to form metal phosphates. The solubility of metal phosphates formed control the bioavailability of that fraction. The combined application of amendments (BALPO) decreased the plant Pb by 55 – 68 % and plant Mn by 40-49 %, when compared to BA. The lowered metal concentration in plants may be due to the less bioavailable fraction, most likely the result of the increase in soil pH by lime application, which ionises pH-dependent exchange sites, raising cation exchange capacity (CEC) and metal sorption to soil particles (Mench et al., 2000). Also compost and phosphate additions lead to the formation of complexes and precipitates, which lower the mobile metal fractions thereby reducing the absorption by plants (Mench et al., 2000). The concentration of Pb in the shoot was three to six times lower than that of the concentration in the root, suggesting a low translocation rate. The retention of Pb in the roots is due to binding to ion exchange sites and extra cellular precipitation, mainly in the form of Pb phosphates, with both these mechanisms occurring in the cell wall (Jarvis and Leung, 2002). Also Pb does not always penetrate the root endoderm and enter the stele since the endoderm acts as a barrier to Pb absorption and penetration to the interior of the stele and its transport to the aerial plant part (Weis and Weis, 2004). Changes in rhizospheric soil properties induced by root exudates (amino acids, sugars, organic acids, peptides, proteins etc) and amendments may have significant influence on the mobility, bioavailability and translocation of trace metals (Arienzo et al., 2003).  7.3.2 Accumulation characteristics of Pb and Mn in plants  ECroot, ECshoot and TF values of Pb and Mn are given in Table 7.3. In the case of Mn, ECroot ranged from 1.4 – 5.9 whereas ECshoot values were from 1.6 – 5.4. For Pb, the corresponding values were 0.13 – 0.49 and 0.05 – 0.25 respectively. Combined application of amendments (BALPO) significantly reduced the ECroot and ECshoot of both Mn and Pb. ECroot and ECshoot for Pb decreased from 0.47 to 0.13 and 0.15 to 0.06, respectively, with the combined addition of amendments (BALPO) in Poa. In the case of Mn, application of lime (BAL) decreased the ECroot and ECshoot, whereas phosphate (BAP) and compost (BAO) application increased the corresponding values. This can be explained by the increased exchangeable Mn fraction in the soil, which received phosphate and compost amendments, in addition to differential metal absorption by plant species due to the influence of variable root exudates released into the rhizosphere. Compared to BA, ECroot and ECshoot for Mn decreased by 60 and 66 % in Poa with  138 the combined application of amendments (BALPO), whereas the corresponding decreases in Lolium were 48 and 43 %. These results again highlight the superiority of combined amendment addition (BALPO) with Poa in stabilising Pb and Lolium in stabilising Mn in the soil.  Table 7.3. ECroot, ECshoot and TF for Pb and Mn  Mn Pb Enrichment Coefficient (EC) Enrichment Coefficient (EC) Plant species Conditions/ Treatments Root Shoot TF Root Shoot TF B0 1.40 1.61 0.94 0.42 0.06 0.47 BA 2.50 2.26 0.91 0.44 0.13 0.29 BAL 2.00 1.75 0.87 0.29 0.06 0.20 BAP 5.40 3.23 0.59 0.25 0.08 0.31 BAO 2.70 2.79 1.03 0.32 0.12 0.38     Lolium BALPO 1.20 1.00 0.80 0.19 0.07 0.35 B0 1.45 1.87 0.67 0.49 0.25 0.51 BA 3.00 2.93 0.97 0.50 0.15 0.29 BAL 2.46 2.06 0.84 0.33 0.06 0.21 BAP 5.94 3.84 0.65 0.24 0.07 0.29 BAO 3.21 3.06 0.95 0.29 0.13 0.42    Festuca BALPO 2.56 1.66 0.65 0.28 0.12 0.36 B0 2.1 1.80 1.61 0.37 0.22 0.59 BA 5.10 5.40 1.04 0.47 0.15 0.3 BAL 2.20 1.66 0.76 0.30 0.05 0.16 BAP 5.70 3.60 0.64 0.26 0.10 0.37 BAO 3.40 3.80 1.12 0.37 0.11 0.3 BALPO 1.98 1.70 0.88 0.13 0.06 0.22     Poa F * * * * * *   Mean values, n = 3. * F significant at P<0.05. B0 – Initial soil, BA – Spiked soil, BAL - Spiked soil plus lime, BAP - Spiked soil plus phosphate, BAO - Spiked soil plus compost, BALPO - Spiked soil plus lime, phosphate and compost.  139 The possible explanation for the superiority may be the ability of plant roots to alter soil conditions, such as pH, organic carbon and soil moisture by root exudation (Susarla et al., 2002) and the soil amendments complementing the plant effect to bring out the assisted natural remediation. The environmental hazards of metal pollution depend on geochemical and biochemical properties of a given metal and are related to several processes taking place in the soil and plants (Mench et al., 2000). The TF (Translocation Factor) for Pb ranged from 0.16 to 0.51 whereas for Mn, it ranged from 0.59 to 1.6 (Table 7.3). For Mn, the lowest TF values were for phosphate amended soils, whereas for Pb, the lowest TF values corresponded to lime amended soil. The lowering of metal transport in plants may be explained by the fact that once they enter the plant, the metals are too insoluble to move freely in the vascular system since they form carbonate or phosphate precipitates immobilizing them in apoplastic (extracellular) and symplastic (intracellular) compartments in the root (Raskin et al., 1997). Also unless the metal ion is transported as a non-cationic metal chelate, apoplastic transport is further limited by the high cation-exchange capacity of cell walls (Raskin et al., 1997).  7.3.3 Pb and Mn fractions in the soils  Pb and Mn fractionation in the soil resulting from plant growth alone is presented in Table 7.4 and by plant growth and amendment addition in Figure 7.1. When the soil was spiked, there was an increase in the exchangeable fraction of both Pb and Mn in soil. The decrease of the mobile fraction was best achieved by the growth of Poa for Pb and Lolium for Mn. Application of amendments had a pronounced effect in further lowering the exchangeable fraction. The maximum decrease was observed in Poa with combined amendments (BALPO) for Pb, and in Lolium with lime amendments (BAL) and combined amendments (BALPO) for Mn. In general, the order of amount of Pb forms in soils with combined addition of amendments (BALPO) was: Pb residual > Pborganic > Pboxide > Pbexchangeable. Addition of lime (BAL) re-distributed >40 % of total Pb to the oxide fraction, whereas addition of phosphate (BAP) re-distributed >62% of total Pb to the residual fraction (mainly bound in silicates). The organic Pb partitioning by compost application was about 32 % of the total Pb in soils grown with Poa. Pb added to soil may react with available soil anions such as SO42-, H2PO41-, HPO42- or CO32- to form sparingly soluble salts and compounds such as lead carbonate Pb 3 (OH) 2 (CO 3 ) 2 and chloropyromorphite (Pb 5 (PO 4 ) 3 Cl) which are least soluble at near  140 neutral pH (Waldron, 1980). This may be the reason for lowering the mobile Pb fraction in soils with the combined amendment addition, since the pH ranged from 6.8 – 7.0 in those soils.  Table 7.4 % partitioning of Pb and Mn in soils with and without plant growth.    % Pb fractions in soil % Mn fractions in soil Treatment /Conditions   Exch Oxide Organic Residual Exch. Oxide Organic Residual B0 2.2 35 22.8 40 11 43.5 11 34.5 BA 4.2 31 25.8 39 19.2 40.5 11.3 29 Soil alone    B0 0.31 38 17 45 7 52 7.1 34 BA 0.51 30 25 45 11 49 19.1 21 Soil + Lolium       B0 0.63 36 23.37 40 9.6 54 8.5 28 BA 0.83 40 21.17 38 15 50 8.2 27 Soil + Festuca     B0 0.14 29.9 24 46 11 45 11.2 33 BA 0.19 42.3 16.51 41 16 44 14 26 Soil + Poa   The major partitioning of each metal in a soil is in bold. n=3. F significant at P<0.05. B0 – Initial soil, BA – Spiked soil.         141     Figure 7.1 Mn and Pb fractionation in soils. B0 – Initial soil, BA – Spiked soil, BAL - Spiked soil plus lime, BAP - Spiked soil plus phosphate, BAO - Spiked soil plus compost, BALPO - Spiked soil plus lime, phosphate and compost.  Distribution of Mn in different forms in soil by the combined addition of amendments (BALPO) was: Mnoxide > Mnresidual > Mnorganic > Mnexchangeable. (Figure 7.1). A similar observation of high affinity of Mn towards the oxide phase was reported by Navas and Lindhorfer (2005). Lime application (BAL) lowered the exchangeable Mn fraction by 40 % while phosphate addition (BAP) increased the exchangeable Mn fraction by 35 %. The combined application of amendments (BALPO) lowered the exchangeable Mn fraction by almost 50 % irrespective of plant species. Soluble, exchangeable and chelated forms are the mobile and bio-available 0% 20% 40% 60% 80% 100% BA L BA P BA O BA LP O BA L BA P BA O BA LP O BA L BA P BA O BA LP O BA L BA P BA O BA LP O Amendments alone Amendments + Lolium Amendments + Festuca Amendments + Poa %  M n  fra ct io n at io n 0% 20% 40% 60% 80% 100% BA L BA P BA O BA LP O BA L BA P BA O BA LP O BA L BA P BA O BA LP O BA L BA P BA O BA LP O Amendments alone Amendments + Lolium Amendments + Festuca Amendments + Poa %  Pb  fra ct io n at io n Exch Oxide Organic Residual  142 fractions in soils (Thangavel. and Subhuram, 2004). Application of phosphates and compost considerably reduced the exchangeable fraction of Pb, whereas for Mn, they had an increasing effect on the mobile Mn fraction. The success of immobilisation by phosphate addition is based on chemical immobilization of the metal contaminant through the formation of insoluble heavy- metal phosphate compounds in the soils, such as pyromorphite for Pb. The immobilisation mechanism is considered to be the dissolution of the Pb compounds followed by the precipitation of Pb phosphate. Thus, successful immobilisation of Pb in soil requires enhancing the solubility of soil Pb and P by decreasing soil pH and applying sufficient phosphorus so as to provide free phosphate ions (H2PO41– and HPO42-) in the soil solution (Ma et al., 1995). By the addition of phosphates in the present study, there was an increase of 35 - 40 % available P in the soil which is obviously the soluble P fraction facilitating the formation of lead phosphates and thereby Pb immobilisation.  Addition of compost (BAO) may lead to complexation of Pb with soil organic matter (humic and fulvic acids), which is then adsorbed onto soil solids. Humic and fulvic acids present in compost are known to enhance the metal adsorption capacity of mineral surfaces through the formation of ternary mineral surface-metal-organic ligand complexes (Arias et al., 2002). These mechanisms may facilitate attachment of substantial amounts of Pb, accounting for the low mobility of lead in soils (Harrison and Laxen, 1981). Even though organic matter is an important reactive component in soils capable of retaining the metal cations and a good sorbent for metals (Ge and Hendershot, 2005), the degree of immobilisation depends on the level of humification of the organic matter. Similar observations of metal immobilisation by increased metal sorption in mineral-humic mixtures were reported in soils with podzolic characteristics due to the acidic soil environment and high organic matter status (Ge and Hendershot, 2005).  Since the aim of the present study was to explore phytoremediation based on mobility/bioavailability of soil metals, the total metal concentrations of soil after amendment treatment and plant growth are less significant, and the observed decrease often within the range of experimental error. Phytostabilisation is not a strategy for removal of metal-contaminants, but it results in in situ inactivation or immobilisation of contaminants. The results from this study show that the exchangeable or mobile fraction of Pb and Mn decrease significantly due to growth of Poa and Lolium in conjunction with combined amendments.   143 7.3.4 Relationships between soil pH and soil metal fractions.    Correlations between soil pH and soil metal fractions   Exchangeable Oxide Organic Residual Pb -0.045 0.363 0.068 0.168 Mn -0.658* 0.707* 0.113 0.125  * R statistically significant at P < 0.05.  Figure 7.2. Relationship between soil pH and metal fractions in soil, (a) soil pH and Pb fractions in the soil (b) soil pH and Mn fractions in the soil.  Correlation studies conducted between different metal fractions and soil pH (Figure 7.2) have shown that the exchangeable fractions of both Pb and Mn are negatively correlated with the pH, whereas other fractions (oxide, organic and residual) are positively correlated. Exchangeable Mn gave a significant negative correlation with soil pH (R = -0.658). A significant positive correlation between pH and oxide fraction was observed for Mn (R = 0.707) whereas for Pb, correlation obtained was not significant (R = 0.363). No significant correlations between pH and organic and residual fractions were observed for either Mn or Pb, indicating that the organic and residual fractions were not significantly affected by soil pH.    0 20 40 60 80 100 120 5 5.5 6 6.5 7 7.5 8 Soil pH Pb   fr a c tio n s  (m g/ kg ) 0 50 100 150 200 250 300 350 5 5.5 6 6.5 7 7.5 8 Soil pH M n   fr a c tio n s  (m g/ kg ) Exch. Oxide Organic Residual (a) (b)  144 7.3.5 Relationships between soil metal concentrations and the Enrichment Coefficient (EC)  Correlation studies conducted between soil metal concentrations and the Enrichment Coefficient (EC) of plants (Table 7.5) revealed that there was a negative correlation between total soil Pb and ECroot and ECshoot for Pb (R = -0.496 and -0.869). In the case of Mn, a moderate and positive correlation was obtained between total soil Mn and ECroot (R = 0.308), while with ECshoot a low correlation was obtained (R = 0.254. This clearly reveals the fact that total metal concentration in the soil cannot be used as a measure to judge the impact of metal contamination on the environment. High metal concentrations in the soil do not always indicate correspondingly high levels of these metals in the plants, since this depends on several factors, such as pH, cation exchange capacity, organic matter, humidity and others (Albasel and Cottenie, 1985).  Table 7.5 Relationship between Enrichment Coefficient (EC) and total soil metal concentration (mg/kg).  Metal Relationship Regression equation Correlation Coefficient (R) ECR & total soil Pb y= -0.269Ln(x) + 1.637 -0.496 Pb ECS & total soil Pb y = -0.173Ln(x) + 0.948 -0.869* ECR & total soil Mn y = 2.468Ln(x) - 11.339 0.308 Mn ECS & total soil Mn y = 0.9685Ln(x) - 3.072 0.254  ECR – Enrichment Coefficient (Root), ECS – Enrichment Coefficient (Shoot) *correlation coefficient significant at P<0.05.                   145 7.3.6 Relationships between soil metal fractions and plant metal concentrations   Figure 7.3 Relationship between plant metals and soil metal fractions. (a) Exchangeable Pb and Root Pb, (b) Oxide Pb and Root Pb, (c) Exchangeable Mn and Root Mn, (d) Exchangeable Mn and Shoot Mn, (e) Oxide Mn and Root Mn, (f) Oxide Mn and Shoot Mn  Correlation studies conducted between soil metal fractions and plant metals (Figure 7.3) revealed that in the case of Pb, there was a positive and significant correlation (P<0.05) between Pbroot and Pbexchangeable in the soil (R = 0.682) and a moderate correlation between Pbroot and Pboxide (R = 0.397). In the case of Mn, there was a significant positive correlation (P<0.05) between Mnexchangeable and Mnplant (both root and shoot) (R values, being 0.589 and 0 20 40 60 80 0.0 0.5 1.0 1.5 Exchangeable  Pb (mg/kg) Ro o t Pb  (m g/ kg ) 0 20 40 60 80 10 30 50 70 Oxide  Pb (mg/kg) Ro o t Pb  (m g/ kg ) 200 600 1000 1400 1800 2200 0 10 20 30 40 50 Exchangeable  Mn (mg/kg) Ro o t M n  (m g/ kg ) 200 600 1000 1400 1800 2200 0 10 20 30 40 50 Exchangeable  Mn (mg/kg) Sh o o t M n  (m g/ kg ) 200 600 1000 1400 1800 2200 100 150 200 250 300 350 Oxide  Mn (mg/kg) Ro o t M n  (m g/ kg ) 200 600 1000 1400 1800 2200 100 150 200 250 300 350 Oxide  Mn (mg/kg) Sh o o t M n  (m g/ kg ) B0 BA BAL BAP BAO BALPO Y = 7.36Ln(x) + 48.64 R = 0.682 Y = 16.69Ln(x) – 18.39 R = 0.397 Y = 232.1Ln(x) + 603.4 R = 0.589 Y = 195.4Ln(x) + 486.5 R = 0.669 Y = 702.8Ln(x) – 2754.2 R = 0.423 Y = 829.03LN(x) – 3530 R = 0.596 (a) (b) (c) (d) (e) (f)  146 0.669 respectively). With Mnoxide also, Mnplant (both root and shoot) gave positive and high correlations (R values, being 0.423 and 0.596 respectively) .  This again confirms the mobility and bioavailability of the exchangeable and oxide fractions of Pb and Mn in the soil. The oxide bound metals are also available for plant uptake (Arias et al., 2002), which may be the result of the low soil pH in the present study. The quantity of metals absorbed by the plant depends on the concentration and speciation of the metal in the soil solution, together with its successive movement from the soil to the root surface and from the root to the aerial part (Patra et al., 2004).  7.3.7 Relationships between soil properties and metal uptake by plants  The influence of soil properties on Pb and Mn uptake (Figure 7.4) by the plants showed that soil pH had a negative effect (P<0.05) on both Pb and Mn uptake (R values being 0.569 and 0.639 respectively). Organic matter status of the soil had a positive effect on both Pb and Mn uptake (R values being 0.365 and 0.511 respectively), whereas available P status of the soil had a positive effect on Mn uptake (R = 0.405) and a negative effect on Pb uptake (R = 0.603). The negative effect of available soil P in reducing soluble Pb in the soil and decreasing Pb concentration in plants has been discussed earlier in the section “Metal concentrations and uptake in plants”. Thus the results obtained clearly reveal that the uptake of metals by plants varies greatly as a function of soil conditions (Patra et al., 2004).  The ability of Pb to bind with organic matter at low soil pH has been reported by Brown et al. (2000) in a study on Pb leaching in peat amended soil. Addition of compost along with lime and phosphate in the present study decreased the uptake of Pb and Mn, which may be the result of more sorption, complexation and precipitation of the metals as a result of the reduction in ion competition with protons in soil at near neutral pH (Yin et al., 1997).       147 Figure 7.4. Relationship between soil properties and metal uptake by the plants. (a)Pb uptake and soil pH, (b) Mn uptake and soil pH, (c) Pb uptake and % organic matter in the soil, (d) Mn uptake and % organic matter in the soil, (e) Pb uptake and available P in the soil, (f) Mn uptake and available P in the soil.                           148    Relationship Regression Equation Correlation Coefficient (R) Soil pH vs Pb uptake y = -49.162Ln(x) + 124.66 -0.569* Soil pH vs Mn uptake y = -4485.2Ln(x) + 9798.7 -0.639* % soil organic matter vs Pb uptake y = 28.686Ln(x) + 14.812 0.365 % soil organic matter vs Mn uptake y = 2719.4Ln(x) – 406.64 0.511 Available soil P vs Pb uptake y = -14.384Ln(x) + 74.57 -0.603* Available soil P vs Mn uptake y = 1019.3Ln(x) – 1215.4 0.405  * R was statistically significant at P < 0.05.  5 15 25 35 45 55 65 5.0 5.5 6.0 6.5 7.0 7.5 8.0 Soil pH Pb  u pt ak e (m ic ro  gr am s/ po t) 0 1000 2000 3000 4000 5.0 5.5 6.0 6.5 7.0 7.5 Soil pH M n  u pt ak e (m ic ro  gr am s/ po t) 5 15 25 35 45 55 65 1.5 2.0 2.5 3.0 % organic matter in the soil Pb  u pt ak e (m ic ro  gr am s/ po t) 0 1000 2000 3000 4000 1.5 2.0 2.5 3.0 % organic matter in the soil M n  u pt ak e (m ic ro  gr am s/ po t) 5 15 25 35 45 55 65 5.0 15.0 25.0 35.0 Available P in the soil (mg/kg) Pb  u pt ak e (m ic ro  gr am s/ po t) 0 1000 2000 3000 4000 5.0 15.0 25.0 35.0 Available P in the soil (mg/kg) M n  u pt ak e (m ic ro  gr am s/ po t) B0 BA BAL BAP BAO BALPO (a) (b) (c) (d) (e) (f)  149 Interaction of organic matter with metals is dependent on metal species and the soil pH. The protonation of negatively charged organic matter and other exchange sites at low pH will make it difficult to coordinate the metals present in soil solution (Ross, 1994).  7.4 Conclusions  • Lowest plant concentrations and uptake values were recorded by Poa for Pb and Lolium for Mn.  • Lime application lowered plant Pb and Mn concentrations whereas phosphate application retarded plant Pb and augmented plant Mn.  • Addition of lime re-distributed >40% of total Pb to the oxide fraction whereas addition of phosphate re-distributed >62% of total Pb to the residual fraction.  • Phosphate addition increased the exchangeable Mn fraction by 35% and the combined application of amendments lowered the exchangeable Mn fraction by almost 50%.  • Compost application increased the Mn concentration (both root and shoot) in Lolium and Festuca, whereas a decreasing effect was noticed for Pb.  • Combined amendment addition resulted in a significant decrease in Pbexchangeable (mobile) in soils growing Poa and Mnexchangeable in soils growing Lolium.  • The re-distribution of the exchangeable fraction was mainly to the oxide fraction for Mn and residual fraction for Pb.  • ECroot and ECshoot for Pb in Poa decreased by 72 and 60% with the combined application of amendments, while the corresponding decreases for Mn in Lolium were 48 and 43%.  • Correlation studies conducted between soil properties and plant metal uptake revealed a positive effect of soil organic matter and available P on Mn uptake and a negative effect on Pb uptake.  This study enabled identification of the best plant-amendment combination that can reduce the mobility and phyto-availability of Pb and Mn in a highway soil, contaminated with Cu, Pb, Mn  150 and Zn due to vehicular traffic. It revealed a cost-effective package for phytostabilisation of Pb and Mn that can not only reduce hazards associated with excess Pb and Mn, but also improve soil quality and restore its functionality.  Thus the environmental hazards of metal pollution depend on the metal–contaminant chemistry influenced by various geochemical and biochemical properties of soil. Application of soil amendments assist natural metal remediation of soil by hastening soil sorption processes and this effect is complemented by growing suitable plant species releasing root exudates with sequestration agents. The refinement of the technique requires molecular studies, which is suggested as the future line of work.                         151 7.5 References  Albasel N. and Cottenie, A. (1985) Heavy metal contamination near major highways, industrial and urban areas in Belgian grassland. Water Air Soil Pollut., 24, 103–110.  Arias, M., Barral, M. T. and Mejuto, J. C. (2002). Enhancement of copper and cadmium adsorption on kaolin by the presence of humic acids. Chemosphere, 48, 1081–1088.  Arienzo, M., Adamo, P. and Cozzolino, V. (2003). The potential of Lolium perenne for revegetation of contaminated soil from a metallurgical site. Science of The Total Environment, 319, 13–25.  Baker, A. J. M., McGrath, S. P., Sidoli, C. M. D. and Reeves, R. D. (1994). The possibility of in situ heavy metal decontamination of polluted soils using crops of metal-accumulating plants. Resour. Conserv. Recy., 11, 41–49.  Brown, P. A., Gill, S. A. and Allen, S. J. (2000). Metal removal from wastewater using peat. Review article. Water Resour., 34, 3907–3916.  Environment Canada. (1987). National inventory of sources and emissions of manganese— 1984; EPS5/MM/1, Conservation and Protection Environmental Analysis Branch, Ottawa  Friedland, A. J. (1990). In Heavy Metal Tolerance in Plants: Evolutionary Aspects, Shaw, A. J., Ed.; CRC Press: Boca Raton, FL, 1990, pp. 7-19.  Ge, Y. and. Hendershot, W. (2005). Modeling sorption of Cd, Hg and Pb in soils by the NICA [non-ideal competitive adsorption]—Donnan model. Soil and Sediment Contamination, 14, 53–69.  Jarvis, M. D. and Leung, D. W. M. (2002). Chelated lead transport in Pinus radiata: an ultrastructural study. Environ. Exp. Bot., 48, 21-32.  Joint FAO/WHO Expert Committee on Food Additives, (2000). Safety Evaluation of Certain Food Additives and Contaminants. WHO Food Additives Series 44, World Health Organization, Geneva.  Harrison, R. M and Laxen, D. P. H. (1981). Lead Pollution: Causes and Control, Chapman Hall, London.  Haeussling, M. and Marschner, H. (1989). Organic and inorganic soil phosphates and acid phosphatase ativity in the rhizosphere of 80-year-old Norway spruce [Picea abies (L.) Karst trees.] trees. Biology and Fertility of Soils, 8, 128-133.  Kumar, P. B. A. N., Dushenkov, V., Motto, H. and Raskin, I. (1995). Phytoextraction: The use of plants to remove heavymetals from soils. Environ. Sci. Technol., 29(5), 1232-1 238.   152 Kumpiene, J., Lagerkvist, A. and Maurice, C. (2007). Stabilization of Pb- and Cu-contaminated soil using coal fly ash and peat. Environmental Pollution, 145, 365-373.  Laidlaw, M. A. S., Mielke, H. W., Filippelli, G. M., Johnson, D. L. and. Gonzalez, C. R. (2005). Seasonality and children's blood lead levels: developing a predictive model using climatic variables and blood lead data from Indianapolis, Indiana, Syracuse, New York and New Orleans, Louisiana (USA). Environ. Health Perspect., 113 (6), 793–800.  Lintern, M. J., Butt, C. R. M. and Scott, K. M. (1997). Gold in vegetation and soil-three case studies from the goldfields of southern Western Australia. J. Geochem. Explor., 58, 1-14.  Ma, Q. Y., Logan, T. J. and Traina, S. J. (1995). Lead immobilization from aqueous solutions and contaminated soils using phosphate rocks. Environ. Sci. Technol., 29, 1118–1126.  Mench, M., Vangronsveld, J., Clijsters, H., Lepp, N. W. and Edwards, R. (2000). In situ metal immobilisation and phytostabilization of contaminated soils. In: T. Norman and G. Banuelos, Editors, Phytoremediation of Contaminated Soil and Water, Lewis Publishers, Boca Raton, FL pp. 323–358.  Ministry of Environment, Lands and Parks. (1995). Criteria for Managing Contaminated Sites in British Columbia. Victoria, British Columbia, Queen’s Printer for B.C.  Navas, A. and Lindhorfer, H. (2005) 'Chemical Partitioning of Fe, Mn, Zn and Cr in Mountain Soils of the Iberian and Pyrenean Ranges (NESpain). Soil and Sediment Contamination, 14(3), 249 – 259.  Needleman, H. L., Schell, A., Bellinger, D., Leviton, A and Allred, E. N. (1990). The long-term effects of exposure to low doses of lead in childhood. An 11-year-follow-up report. N. Engl. J. Med., 322, 83-88.  Padmavathiamma, P. K. and Li, L. Y. (2007). Phytoremediation Technology: Hyper- accumulation metals in plants. Water, Air, and Soil Pollution, 184, 105-126.  Padmavathiamma, P. K. and Li, L. Y. (2009). Phytoremediation of metal-contaminated soil in temperate humid regions of British Columbia, Canada. International Journal of Phytoremediation, 11(6), 575-590.  Patra, M., Bhowmik, N., Bandopadhyay, B. and Sharma, A. (2004). Comparison of mercury, lead and arsenic with respect to genotoxic effects on plant systems and the development of genetic tolerance. Environ. Exp. Bot., 52, 199-223.  Preciado, H. F. and Li, L. Y. (2006). Evaluation of metal loading and bioavailability in air, water and soil along two highways of British Columbia, Canada. Water, Air, and Soil Pollution, 172, 81–108.  Raskin, I., Smith, R. D. and Salt, D. E. (1997). Phytoremediation of metals: using plants to remove pollutants from the environment. Curr. Opin. Biotechnol., 8, 221–226.  153  Ross, S. M. (1994). Retention, transformation and mobility of toxic metals in soils. In: S.M. Ross, Editor, Toxic metals in soil-plant systems, John Wiley and Sons, Chichester. pp. 63– 152.  Sezgin, N., Ozcan, H. K., Demir, G., Nemlioglu,S. and Bayat, C. (2003). Determination of HeavyMetal Concentrations in Street Dusts in IstanbulE-5 Highway Environment International, 29, 979-985.  Simon, L. (2005). Stabilization of metals in acidic mine spoil with amendments and red fescue (Festuca rubra L.) growth. Environ. Geochem. Health, 27, 289–300.  Smith, R. A. H. and Bradshaw, A. D. (1979). The use of metal tolerant plant populations for the reclamation of metalliferous wastes. Journal of Applied Ecology, 16, 595–612.  Susarla, S., Medina, V. F. and McCutcheon, S. C. (2002). Phytoremediation: an ecological solution to organic chemical contamination. Ecol. Eng., 18, 647–658.  Tessier, A., Cambell, P.G.C. and Bisson, M.(1979). Sequential extraction procedure for the speciation of particulate trace metals. Anal. Chim., 51, 844–851.  Thangavel, P. and Subhuram, C. V. (2004). Phytoextraction - Role of hyper accumulators in metal contaminated soils. Proc. Indian Natn. Sci. Acad., B, 70 (1), 109-130.  US Environmental Protection Agency. Health effects support document for manganese. (2003). Available from: www.epa.gov/safewater/ccl/pdf/manganese.pdf. Accessed 1/10/04.  Yin, Y., Allen, H. E., Huang, C. P. and Sanders, P. F. (1997). Interaction of Hg(II) with soil- derived humic substances. Analytica Chimica Acta, 341, 73.  Waldron, H. A.(1980). Metals in the environment, Academic Press, London (NY).  Weis, J. S. and Weis, P. (2004). Metal uptake, transport and release by wetland plants: implications for phytoremediation and restoration. Environ. Int., 30, 685-700.    154 8. 7RHIZOSPHERE INFLUENCE AND SEASONAL IMPACT ON PHYTOSTABILISATION OF METALS – A FIELD STUDY  8.1 Introduction  A wide range of pollutants such as particulates, trace elements and petroleum hydrocarbons, which originate from transportation activities, accumulate on highway surfaces in addition to direct aerial deposition. Due to the impermeability of the pavement, these pollutants are delivered by highway runoff during wet weather to roadside soils and potentially into streams via drainage networks (Thomson et al., 1997) and enter the food chain (Ma and Jennings, 2008). Of all the contaminants in the highway system, metals are the most prevalent contaminants of concern (Hathhorn and Yonge, 1996), since they are adsorbed on the soil exchange complex. High mobile concentrations lead to ecological and human health risks, as metals leach into receiving waters and enter the food chain (Alloway, 1990; Kabata-Pendias and Pendias, 2001). Highway runoff contains high concentrations of lead, zinc, copper and manganese resulting from wear of brakes, tires, and other vehicle parts and due to the introduction of methyl cyclopentadienyl manganese tricarbonyl (MMT) as a replacement for tetra-ethyl lead in 1974 (FHWA, 1998; Hall et al., 1998). While leaded gasoline has been suspended in North America and most of industrialized countries for more than three decades, residual Pb remains a concern (Hodes et al., 2003). Land associated with 12,000 km of roads in B.C. (and millions of kilometres around the world) endanger wildlife habitats where metal contamination needs to be remediated in a comprehensive manner (Precciado and Li, 2006). Environmental management of metal contamination is a challenging geotechnical and ecological problem. Currently 45 of the 50 US states and 8 of the 13 Canadian provinces and territories provide regulatory guidance on the maximum metal concentrations in surface soils, which do not yield unacceptable human health risk (Jennings and Petersen, 2006).     7  A version of this chapter will be submitted for publication. Padmavathiamma, P.K. and Li, L.Y. (2009). Rhizosphere influence and seasonal impact on phytostabilisation of metals – a field study   155 Previous studies have dealt with the identification of plant species for phytoremediation and suitabilities of different plant amendment combinations on the immobilisation of metals in soil (Padmavathiamma and Li, 2009a, b). However, verification of results under field situations is necessary. The mobile-immobile distributions of metal fractions, controlled by various pedogenic and biogenic processes, are influenced by seasonal changes (Kim and Fergusson, 1994, Duman et al., 2006) and metal-rhizosphere interactions (Jacynthe, 2007). Hence comprehensive studies are conducted under field conditions, taking into account seasonal and rhizosphere influences on soil-plant-metal contaminant chemistry, to develop Best Management Practices (BMP) for phytostabilisation of metals. The main goal of this study was field verification of the results obtained from previous pot experiments.  The study was undertaken in a Luvic Gleysolic soil, Deltaport Way along HW 17 NB ramp in Delta, BC using soil amendments of lime and phosphate in combination with three previously identified plant species, Lolium perenne, Festuca rubra and Poa pratensis (Padmavathiamma and Li, 2007). Four metals, Cu, Pb, Mn and Zn were studied and treatment efficiencies were assessed during three seasons - summer, autumn and winter. The research tasks included (1) quantifying the seasonal extent of metal accumulation in soil and assessing the seasonal impact on metal speciation in the soil as influenced by amendments and different plant species; (2) determining metal accumulation in different plant parts seasonally; and (3) assessing the influence of root-soil interactions on metal dynamics. The final outcome of the study is the development of a remediation strategy for four metals (Cu, Pb, Mn and Zn) involving suitable plants and amendments, taking into account of seasonal and rhizosphere influences, while maintaining the functional and biological integrity of soil after remediation.  8.2. Materials and methods  8.2.1 Experiment details  The study area was located on the northern ramp of HW 17, Deltaport Way, Delta, B.C. Experimental details are given in Table 1. The study was conducted as a Completely Randomized Factorial Experiment in Split plot design (Appendix D, Figure 1) with three replications. The three plant species tested individually and in combination were Lolium perenne, Poa pratensis and Festuca rubra. The amendments were lime and phosphate, selected  156 on the basis of the effect of lime on soil pH which controls metal solubility/mobility and the effect of P as a metal retardant in soils (Padmavathiamma and Li, 2008). 24 stratified randomly selected plots (1 m2) were laid out along a 100 m transect at distances of 5, 9, 11, 14 and 17 m from the edge of the ditch. The surface vegetation was removed, the soil was loosened to a depth of 15 cm and amendments incorporated with the soil.  Table 8.1 Experimental program for field study Heavy metals studied Conditions/Treatments Plant species Stages of sampling  Cu   Pb   Mn   Zn  Soil alone (T0)   Soil plus amendments (lime and phosphate) (T1). Lime (10 tons/ha), Phosphate (135 kg P2O5/ha). Sources of lime and phosphate are dolomite (finely ground) and Ca HPO4. 2H2O.  Loium perenne   Poa pratensis   Festuca rubra   Combination (L. perenne + P. pratensis+F. rubra)  90 DAS (days after sowing)   180 DAS (days after sowing)   270 DAS (days after sowing). Design –Completely Randomized Factorial Experiment in Split Plot Design. 8 treatment combinations and three replications. Seeds sown in May 2007 and the soil and plant samples collected during three different seasons, i.e. 90 DAS, August 07 (summer), 180 DAS, November 07 (autumn) and 270 DAS, February 2008 (winter).  8.2.2 Collection of samples and laboratory analysis  The field soil was analysed for its basic physico-chemical characteristics, including pH, electrical conductivity, CEC, particle size distribution, % carbon, % nitrogen and total metal concentrations. After planting, soil samples collected during different seasons (summer, autumn and winter) were analysed for pH, electrical conductivity and total metal concentrations. During winter both rhizosphere soil (RS) and bulk soil (BS) were collected. Rhizosphere soil (RS) was removed from the roots with gentle shaking, whereas bulk soil (BS) was the soil outside the planted area in the plot where no roots are found (Gobran, et al., 2001). The metal fractionation in the soil for different seasons was estimated by the SSE procedure proposed by Tessier et al. (1979). For the plant samples, shoots and roots were separated, processed, ashed and analysed for their metal content (Padmavathiamma and Li, 2009a). Soil as well as plant extracts were analyzed for Cu, Pb, Mn and Zn using a Varian Spectre AA 220 Multi-element Fast Sequential  157 Atomic Absorption Spectrometer. Quality checks and calibrations were performed using blanks, duplicate samples and reference materials.  8.2.3 Statistical analysis  Statistical analysis was conducted using SAS version 9.1 (SAS Institute, 2001). The statistical significance of differences among means was determined by Analysis of variance (ANOVA) to compare the treatment effects on soil metal speciation, total soil metal concentration, as well as metal uptake by plants. To assess the accumulation characteristics and translocation properties of metals in plants, translocation factor (TF) and enrichment coefficient (EC) were determined. EC of root (Croots/Csoil, the ratio of root to soil concentration) and shoot (Cshoots/Csoil, ratio of shoot to soil concentration) and TF (Cshoots/Croots, ratio of shoot to root concentration) were calculated (Mattina et al., 2003; Kumar et al., 1995).  8.3. Results and discussion  The basic characteristics of the soil are given in (Table 8.2). The texture of the soil is silty clay loam and the taxonomic name of the soil according to the Canadian System of Soil Classification is Humic Luvic Gleysol.  Table 8.2 Key soil characteristics before the field experiment Parameters Values Soil pH 5.64 Electrical Conductivity (dS/m) 0.75 % organic matter 4.5 Total nitrogen (%) 0.30 Available phosphorus (mg/kg) 6.1 Cation exchange capacity (molc kg-1) 22 Total metal concentrations (mg/kg) Cu – 65, Pb – 98, Mn – 210, Zn - 175 Each value represents mean of three measurements.   158 8.3.1 pH and Electrical Conductivity  pH and electrical conductivity (dS/m) in soils growing different plant species, with and without treatments during three seasons are given in Figure 8.1. The lower pH values, during summer may be due to oxidation of metal salts as a result of increased aeration of the soils and removal of nutrient base cations. Release of CO2 from root respiration may reduce pH of rhizosphere soil. However, there was an increase of soil pH at 270 DAS, ie. during winter. The soil pH, initially 5.6, increased to 6.0 in Lolium growing soils, 5.8 in Festuca growing soils, 5.9 in Poa growing soils and 6.2 in soils growing a combination of these plants (1/3 each), at 270 DAS, i.e. winter. The increase in soil pH during winter may be attributed to the dilution of soil solution by higher precipitation (see Appendix D, Table 1) during the winter. When a soil solution is diluted, the concentration of H+ ions becomes diluted, causing higher pH (Jackson, 1958). Furthermore, pH changes by plants may be due to the imbalance in cation or anion release, caused by excess anion or cation uptake (Marschner, 1995). In plots where lime and phosphate amendments were applied (T1), an increase in soil pH was noticed compared to T0 (without amendments),  Figure 8.1.   159 Lolium Festuca Poa Combination   Figure 8.1 Seasonal influences on pH and electrical conductivity of soil. T0 – without amendments, T1 – with amendments (lime plus phosphate).  Soil pH is an important parameter affecting the solubility and mobility of metal fractions, and hence ecological and human-health risks (Sreevastava and Guptha, 1996). Soil pH, sometimes called the “master variable”, has the potential to modify metal solubility/availability in several ways (McBride, 1994), including dissolution/precipitation reactions, regulating the ionisation of pH dependent exchange sites on organic matter and oxide clay minerals and influencing metal speciation in soils (Adriano et al., 2004; Sparks, 2003; Conesa et al., 2006). Chemical forms of metals in soil depend on both the source of contamination and the physicochemical properties of the soil such as pH, Eh, %organic matter and %clay. The electrical conductivity generally increased in summer, both in T0  and  T1 (Figure 8.1), possibly due to the result of lower precipitation and higher evapotranspiration during this season.  8.3.2 Metal concentrations in soil  The average total metal concentrations in the soil before plant growth were 65 mg/kg for Cu, 98 mg/kg for Pb, 210 mg/kg for Mn and 175 mg/kg for Zn. Summer had an augmenting effect on 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 Summer Autumn Winter So il pH 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 Summer Autumn Winter So il pH 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Summer Autumn Winter El e c tr ic a l C o n du c tiv ity  (dS /m ) 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Summer Autumn Winter El e c tr ic a l C o n du c tiv ity  (dS /m ) (T0) (T1) (T0) (T1)  160 total metal concentrations, whereas autumn and winter had a retarding effect. However, total metal concentrations cannot be used to predict the impact of metals on the environment since only the mobile and soluble metal fraction has the potential to leach or to be taken up by plants and enter the food chain (Simon, 2005, Kumpiene et al., 2007; Mench et al., 2000). Since the bioavailability and mobility of metals depend on their association with various soil components, it is essential to quantify the metal bound to each fraction in the soil (Chopin et al., 2008).  8.3.3 Metal fractionation in soil  The fractionation of metals (%) in the soil as influenced by growth of plants, application of amendments and influence of seasons are discussed separately. The results are given in Figure 8.2. The metal fractionation in soils before plant growth is given in Table 8.3.  Table 8.3. % Metal fractionation in the soil before plant growth  % Metal fractionation Metal Exch. Carbonate Oxide Organic Residual Cu 10 5 19 32 34 Pb 4 2 10 42 42 Mn 12 1 42 6 39 Zn 12 5 34 16 33  Mean values. n = 3.  Before plant growth, Cu and Pb were mainly partitioned into organic and residual fractions, whereas Mn and Zn were in the exchangeable and oxide fractions. High organic matter content of the soil (4.5%) and the affinity of Cu and Pb to complex with organic ligands (Sanders et al., 1986; Ross, 1994) explain the large partitioning of Cu and Pb in the organic form. The presence of fresh organic matter can increase metal solubility, due to the availability of soluble organic compounds which form complexes with the metals (Almas et al., 1999; Shuman, 1999), whereas the humic substances in soil organic matter can reduce metal solubility by forming stable metal chelates ( Ross, 1994). Hence the extent of humification of soil organic matter governs the mobility or immobility of organic metal chelates. Carbonate fraction is unlikely in soils with pH 5.6. Extractants used in SSE are not 100% specific and there are chances of extracting non-targeted metal fractions.  161  The influence of plant growth on soil metal fractionation is given in Figure.8.2. 94% of the total Pb was found to exist in the organic, oxide and residual fractions and <5 % in the exchangeable fraction. The exchangeable fraction was significantly lowered (P <0.05) in Festuca growing soils for Cu, Lolium growing soils for Mn, and the combination (Lolium + Poa + Festuca) for Pb and Zn. This explains the suitability of the above plant species to reduce the mobile fractions of each metal studied. These results are in agreement with the previous studies with pot experiments (Padmavathiamma and Li, 2009a, b), except that the plant combination was found to be superior for reducing the exchangeable Pb and Zn under field conditions, in contrast to Poa in the pot experiments. A significant increase of the oxide fraction (P <0.05) in soils growing Festuca was observed in the case of Cu and Mn (Figure 8.3). However, in general, the oxide fraction dominated in Festuca growing soils and organic fractions in soils growing Poa and Lolium during all seasons. This may be explained by the well-developed fibrous root system of Poa and Lolium contributing to increased organic matter of soil (Padmavathiamma and Li, 2008). The differences between plant species in re-distributing metal fractions may be their inherent mechanism of responding to the soil metals by adsorption onto the root surface, absorption and accumulation in roots or precipitation within the root rhizosphere by release of root exudates (Marschner et al., 2007). The adsorption onto the root surface may be by binding of metal cations to the carboxyl functional groups on the root cell walls of the grass species.  The effect of amendment application on metal fractionation in soils with different plants is shown in Figure 8.2. Application of amendments (lime plus phosphate) decreased the metalexchangeable and increased the metalcarbonate fraction, especially for Cu and Pb. This may be due to the effect of amendments on the pH of the soil.          162  Figure .8.2 % metal fractionation in soil by the influence of plants, amendments and seasons. (a) Cu, (b) Pb, (c) Mn, (d) Zn. T0 – without amendments, T1 – with amendments. L – Lolium, F- Festuca, P – Poa, C – Combination (Lolium + Festuca + Poa). FS – Fallow soil. Mean values, n = 3. F significant at P<0.05 for all the four metals. SE (Standard Error) of means given in Appendix E.    163  0% 20% 40% 60% 80% 100% T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 L F P C FS L F P C FS L F P C FS Summer Autumn Winter C u  fra c tio n s  (% ) 0% 20% 40% 60% 80% 100% T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 L F P C FS L F P C FS L F P C FS Summer Autumn Winter Pb   fra c tio n s  (% ) 0% 20% 40% 60% 80% 100% T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 L F P C FS L F P C FS L F P C FS Summer Autumn Winter M n   fra ct io n s  (% ) 0% 20% 40% 60% 80% 100% T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 L F P C FS L F P C FS L F P C FS Summer Autumn Winter Zn   fra ct io n s  (% ) Exch. Carbonate Oxide Organic Residual (a) (b) (c) (d)  164 Cu is less affected by changes in pH (Berggren, 1989 and Strobel et al., 2001) compared to other metals such as Zn, Mn and Pb. There are reports (Brezonik et al., 2003) that Cu is not mobilised by soil acidification. However, the ability of Cu to form chelates with organic compounds (Strobel et al., 2001) may affect the solubility of Cu, as the organic matter content of the soil is about 4.5%. The mineralization and humification processes of these compounds with time led to the fixation of Cu in the soil, converting Cu into insoluble forms. Cu has high affinity for peat moss, humin and humic acids compared to other metals and hence is more liable to form metal organic complexes, which may become unavailable at high pH (de la Rosa et al., 2003). Thus Cu when complexed by humic substances is immobile and less affected by changes in soil pH than the other three metals investigated.  The carbonate bound fraction was higher for Cu and Pb, than for Mn and Zn (Figure 8.2). The relative partitioning of metals in oxide and residual fractions of soil was higher in T1 than in T0 plots. Re-distribution in relatively immobile fractions may be due to various sorption processes: adsorption to mineral surfaces, formation of stable complexes with organic ligands, surface precipitation and ion exchange (Mench et al., 2000; Kumpiene et al., 2007). Applications of lime and phosphate amendments influence pH, redox potential, electrical conductivity and cation exchange capacity which govern sorption/dissorption, precipitation/dissolution and speciation/complexation in the soil (Kumpiene et al., 2007). The enhancement of soil pH in amended soils helps to explain the decrease in metal-exchangeable (mobile metal fractions) in soils that received amendments. Phosphate enhances the immobilization of metals in soils through various processes including direct metal adsorption by phosphate, phosphate anion- induced metal adsorption, and precipitation of metals with solution phosphate as metal phosphates (Adriano et al., 2004). Precipitation as metal phosphates has been shown to be one of the main mechanisms governing the immobilization of metals, such as Pb and Zn, in soils (McGowen et al., 2001). These relatively stable metal-phosphate compounds have extremely low solubility over a wide pH range, rendering phosphate application an attractive technology for managing metal-contaminated soils (Adriano et al., 2004).  The seasonal influence of metal partitioning is evident from Figure 8.2. With the growth of the plants, the exchangeable Cu, Pb and Zn were highest in summer, declining in autumn and  165 winter. Exchangeable Mn was highest in autumn, followed by winter and summer. The availability of Mn in soil depends on its pH and oxidation-reduction potential. Reduced forms of Mn are more bio-available (Marschner, 1988). Mn can occur in more than one valence state, and the more oxidized state precipitates by the formation of hydroxides (or hydrous oxides). Up to 6.0, the hydroxides will not precipitate and solubility increases (Brady and Weil, 1996). During summer and autumn, Cu and Pb occur mainly in the carbonate, oxide and organic fractions, whereas Mn and Zn are likely to be found in the oxide and exchangeable fractions. During winter, partitioning was mainly in the oxide and residual fractions for all four metals (Cu, Mn, Pb and Zn). The re-distribution from the organic fraction to the oxide and residual fraction during winter leads to the possible formation of soluble organic metal chelates during the summer, either absorbed by the soil biota or leached to the surrounding water ecosystem (Almas et al., 1999; Shuman, 1999). During winter almost all of the metalexchangeable and metalcarbonate partitioned into the organic, oxide and residual forms (Figure 8.2). Re-distribution to the immobile fractions during winter led to decreased bioavailabilty, thereby reducing the risk of contaminant transfer and accumulation into the food chain (Kabata-Pendias and Pendias, 2001; Lee et al., 2003). Thus the bioavailable fraction (otherwise known as the mobile or labile fraction) emerges as a relevant factor in risk assessment and environmental monitoring (Adriano et al., 2004). The mobile fractions of metals, i.e. metalexchangeable and metalcarbonate were significantly reduced in soils growing Festuca for Cu, Lolium for Mn and a combination of Lolium + Poa + Festuca for Pb and Zn. Application of amendments (lime + phosphate) decreased the metalexchangeable for all four metals. During winter, the major partitioning changed to the oxide and residual fractions for each of these metals, leading to decreased bioavailability, reducing the risks of contaminant transfer and accumulation into the food chain.  As stated above, remediation methods applicable to soils contaminated with metals are based on two approaches: removal/extraction of the metals from the matrix or reduction of metal mobility or bio-availability in soil (Smith and Bradshaw, 1979; Simon, 2005). When metalexchangeable is decreased in soil, the solubility or bioavailability of metals is reduced, minimising the impact on soil organisms and plants, thereby reducing the exposure pathways (Simon, 2005; Mench et al. 2000). The bioavailability of metallic ions not only depends on the solubility, but also on the thermodynamic activity of the uncomplexed ion (Petrangeli et al. 2001).      166 8.3.4 Metal concentrations and uptake in plants  Metal concentration, expressed as accumulation per unit weight (mg/kg dry weight), and metal content, which denotes the total uptake or removal/m2 are discussed separately. Metal concentrations in different plant species, with and without application of amendments for three seasons are provided in Table 8.4, whereas the metal uptake by plants is given in Figure 8.3. The supply of ions to plants is controlled by the kinetics of solubilisation of ions adsorbed to the solid phase of soil (Chaney et al. 1997). The metal concentrations in plants (mg/kg) with and without application of soil amendments during summer, autumn and winter are given in Table 8.4.  Without application of amendments (T0), the lowest metal concentrations were as follows: Lolium for Cu and Mn, and the combination for Pb and Zn. With the application of amendments (T1), Festuca recorded the lowest concentrations for Cu, Lolium for Mn and the combination for Pb and Zn (Table 8.4). The same trend was found for the metal uptake, except for Zn, for which Poa gave the lowest value in T1 plots. Irrespective of plant species, the root concentration was higher than shoot concentration for all four metals studied. This is consistent with the findings from previous pot experiments (Padmavathiamma and Li, 2008). The partitioning of metals into various soil chemical pools by plant growth (Figure.8.2) is reflected in the plant metal concentrations, in agreement with reports by Brooks (1998) and Weis and Weis (2004) that plant growth significantly influences metal speciation in soil, contributing to the mobility or phyto-availability of metals.             167 Table. 8.4. Metal concentrations in plants (mg/kg), (n = 3, mean values ± S.D). F significant at P <0.05 Summer Autumn Winter   Metal  Plant species Root Shoot Root Shoot Root Shoot Without application of amendments (T0)   Lolium 51±10 24±4 57±16 16±5 66±4 11±3 Cu Festuca 86±15 26±8 78±17 28±4 89±10 23±7.   Poa 66±21 23±12 62±12 20±7 77±14 16±5   Combination 61±9 28±8 58±18 24±10 69±11 20±7    Lolium 24±4 9±1 28±3 13±3 31±7. 6±4 Pb Festuca 19±6 7±1 20±4 10±4 25±3 5±2   Poa 17±8 5±2 19±2 9±0.6 26±9 4±1   Combination 12±7 4±3 13±6 7±0.9 20±3 3±0.4    Lolium 65±28 35±11 99±30 73±9 72±15 62±18 Mn Festuca 87±18 76±9 104±21 87±11 89±22 54±18   Poa 112±40 104±17 123±17 111±10 134±23 82±23   Combination 99±34 92±13 118±18 97±20 124±28 79±19    Lolium 63±15 50±12 68±5 39±5 71±6 31±8 Zn Festuca 69±11 51±9 75±9 41±4 79±13 29±13   Poa 47±11 38±7 58±13 32±12 66±11 25±4   Combination 40±13 30±8 48±11 25±10 57±14 19±13 With application of amendments (T1)   Lolium 47±14 21±2 51±8 18±6 58±8.8 16±2.5 Cu Festuca 40±8 18±4 47±16 16±3 51±13 13±4   Poa 54±16 23±11 58±2 20±8 61±6.7 17±4.9   Combination 56±8 26±3 51±7 24±6 59±9.9 19±4.7    Lolium 16±3 7.8±0.1 17±3 10±1.9 19±3 3±0.4 Pb Festuca 15±6 6.9±2 22±10 9±0.9 22±6 5.3±0.8   Poa 13±7 3.7±0.9 14.9±7 7±0.3 16±4 2.3±0.3   Combination 9±3 2.3±0.6 13.5±8 5±0.6 18±8 1.9±0.2    Lolium 49±20 38±14 69±13 59±13 97±19 35±6 Mn Festuca 67±11 57±12 78±15 66±10 102±16 42±16   Poa 101±23 89±20 112±13 109±18 121±15 79±21   Combination 89±12 82±5 104±15 84±9 110±19 66±13    Lolium 56±12 42±15 62±13 33±12 69±8 27±6 Zn Festuca 61±13 46±9 70±11 40±11 73±4 30±7   Poa 36±6 28±7 49±8 29±6 58±2 20±3   Combination 29±9 25±7 40±10 20±9 51±14 16±11   168 Metal uptake by plants during three seasons is portrayed in Figure 8.3. The shoot uptake was highest in summer for Cu and Zn, whereas it was highest in autumn for Pb and Mn. The root uptake was found to be highest during winter for all the four metals studied. The same trend, shown for metal concentration (Table 8.4) and uptake (Figure 8.3), reveals that plant biomass did not contribute to the accumulation of metals in the plants. During winter, the shoot concentration for Cu was only 1/6th of the root concentration, and for Pb it was only about 1/10th. Variations in the seasonal pattern of uptake potential of metals may be due to the growth effect and other factors such as the quantity of rainfall, abiotic factors such as temperature and variations in metal concentrations in the environment. The lower Mn uptake during summer may be due to the increase in Mn-oxidizing bacteria in the rhizosphere (Arines et al., 1992), which may reduce the oxidation-reduction potential and availability of Mn in the rhizosphere. The changes in plant metal concentrations and uptake may be due to the growth effect, changes in metal concentrations in the environment during different seasons as well as abiotic factors such as temperature, precipitation etc.                    169 Figure 8.3. Metal uptake by the plants during three seasons. (a) – Cu uptake, (b) – Pb uptake, (c) – Mn uptake, (d) – Zn uptake. Mean values. n = 3, F significant at P <0.05. L- Lolium, F – Festuca, P – Poa, C- Combination (.Lolium + Festuca + Poa). S – summer, A – autumn, W – winter. T0 – without amendments, T1 – with amendments.                             170  0 200 400 600 800 1000 1200 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 L F P C L F P C L F P C S A W Cu  u pt ak e (m ic ro  gr am s/ sq . m ) 0 50 100 150 200 250 300 350 400 450 500 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 L F P C L F P C L F P C S A W Pb   u pt ak e (m ic ro  gr am s/ sq . m ) 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 L F P C L F P C L F P C S A W M n   u pt ak e (m ic ro  gr am s/ sq . m ) 0 500 1000 1500 2000 2500 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 T0 T1 L F P C L F P C L F P C S A W Zn    u pt ak e (m ic ro  gr am s/ sq . m ) Root Shoot (a) (b) (c) (d)  171 Even though the shoot uptake was less than the root uptake for all four metals studied, the % translocation to the above-ground portions was higher for Mn and Zn than for Cu and Pb. Lime and phosphate amendments decreased both root and shoot uptake of all four metals. These soil amendments influence key processes and factors that control the dynamics of trace elements in soil, thereby assisting natural attenuation of metal contaminants (Adriano et al., 2004). These changes in soil properties modify soil processes, resulting in mobilisation or immobilisation and regulating the entry of metals into the plants. Stable metal-phosphate compounds formed by the application of phosphate amendment, especially in the case of Pb and Zn, have extremely low solubility over a wide pH range, reducing the entry of metals into the plants in metal- contaminated soils (Adriano et al., 2004; Simon, 2005). The uptake of Cu, Mn, Pb and Zn by plants is often found to decrease with liming, which is attributed to the increased adsorption/precipitation at high pH (Geebelen et al., 2002; Cox and Rains, 1972).             There are differing reports with respect to metal accumulation in plants during different seasons. Some scientists (Kim and Fergusson, 1994; Brekken and Steinnes, 2004) have stated highest metal contents (Cd, Cu, Ni, Pb, Sn, Zn) during autumn and relatively low levels during the spring, whereas others (Wilkins, 1978; Martin and Coughtrey, 1982) have indicated the highest foliar levels during spring and the lowest during winter. Cacador et al. (2000) observed more heavy metal accumulation in Spartina maritima and Halimione portulacoides in summer. Djingova and Kuleff (1994) established that the developmental stage was the most significant explanatory factor for heavy metal accumulation in shoots, since it may be the stage of maximum biomass production.  8.3.5 Metal accumulation charecteristics in plants  The metal accumulation pattern in plants as explained by EC (Enrichment Coefficient) for roots and shoots and TF (Translocation factor) are given in Table 8.5. The seasonal influence on the accumulation pattern is given in Figure 8.4.       172 Table 8.5.  Enrichment Coefficient (ECroot and ECshoot) and Translocation Factor (TF) of metals with and without amendments  without amendments (T0) with amendments (T1)  Metal  Plant species ECR ECS TF ECR ECS TF Lolium 0.74 0.36 0.41 0.55 0.26 0.35 Poa 0.54 0.29 0.45 0.84 0.28 0.34 Festuca 1.12 0.38 0.44 0.49 0.21 0.31  Cu    Combination 0.66 0.32 0.43 0.54 0.23 0.42  F   * NS NS * NS * Lolium 0.24 0.06 0.23 0.2 0.05 0.21 Poa 0.2 0.05 0.22 0.15 0.04 0.14 Festuca 0.21 0.04 0.19 0.22 0.02 0.19  Pb    Combination 0.15 0.04 0.25 0.11 0.03 0.17  F   * NS NS * NS * Lolium 1.23 1.17 0.78 1.1 1.01 0.53 Poa 2.63 1.38 0.92 1.58 1.29 0.78 Festuca 1.27 1.13 0.91 1.13 1.07 0.88  Mn    Combination 1.61 1.42 0.91 1.25 1.17 0.8  F   * * * * * * Lolium 0.68 0.42 0.77 0.45 0.38 0.71 Poa 0.51 0.29 0.70 0.40 0.25 0.63 Festuca 0.71 0.49 0.78 0.59 0.45 0.73  Zn    Combination 0.44 0.27 0.69 0.41 0.26 0.61 F  * * NS * * *  Mean values, n = 3. * F significant at P<0.05. NS – not significant.   From EC and TF values (Table 8.5), it is observed that ECshoot and TF were significantly decreased (P<0.05) by the application of lime and phosphate amendments. This suggests that lime not only increased the soil pH, favouring the sorption of metals in the soil, but also inhibited the translocation of metals, especially Pb from root to shoot (Basta and Tabatabai, 1992). The lowest ECR, ECS and TF values were recorded in Festuca for Cu, Lolium for Mn and Combination for Pb and Zn (Table 8.5).  173   0 0.5 1 1.5 2 2.5 3 ECR ECS TF ECR ECS TF ECR ECS TF Summer Autumn Winter M et al  co n ce n tr at io n  ra tio Cu Pb Mn Zn   Figure 8.4. ECR (Enrichment Coefficientroot), ECS (Enrichment Coefficientshoot), TF (Translocation Factor) of metals during different seasons. Mean values, n = 3. F significant at P<0.05 for all the four metals.  Application of amendments (lime plus phosphate) significantly reduced the EC and TF. ECshoot was highest during summer for Cu and Zn, but during autumn for Pb and Mn (Figure 8.4). ECroot was highest in winter for all four metals. TF of each of these metals was lowest during winter (Figure 8.4), indicating that there was minimum transport of metals to the above-ground portion, with enrichment of metals in the roots. The translocation of Cu and Pb to the above-ground portion was only 20-30% of the root accumulation, whereas for Mn and Zn, it was 60 – 80 %of the root accumulation.  The ultimate objective of a remediation process must be not only to remove/immobilise the metal pollutants from the soil, but most important, to sustain soil health, i.e. the continued capacity of soil to function as a vital living system, sustaining biological productivity, promoting the quality of air and water, and maintaining plant, animal, and human health (Doran and Safley, 1997). In this context, using natural amendments and plant species to accelerate natural soil attenuation/remediation has an edge over other remediation strategies. In general, the EC and TF values should be <1 for phytostabilisation and >1 for phytoextraction (Brooks 1998). In the present study, the EC and TF of studied plants were <1 for all metals except Mn. Even if  174 phytostabilisation cannot be considered as a clean-up method, this study indicates that it can reduce the inherent risk associated with a contaminated site based on a reduction of the soil mobile and bioavailable metal fraction.  8.3.6 Bulk soil (BS) vs Rhizosphere soil (RS)  8.3.6.1 pH  Soil pH was found to be higher and electrical conductivity lower in the bulk soil (BS) compared to those in the rhizosphere soil (RS) (Figure 8.5).  5.4 5.6 5.8 6 6.2 6.4 L F P C So il pH Rhizosphere soil Bulk soil  Figure 8.5  pH of Rhizosphere soil and Bulk soil. L – Lolium, F – Festuca, P- Poa, C - Combination. Mean values, n = 3. F significant at P<0.05. Data set is the mean of T0 and T1. SE of mean = 0.45.  The difference in soil pH between BS and RS may be due to root exudation, microbial respiration and unequal uptake of cations and anions by plant roots (Lombi et al., 2001). Since plants absorb most mineral nutrients and metals as ions, imbalances in the absorption of cations and anions result in root excretion of compensating H+, OH- and HCO3- ions into the rhizosphere causing pH changes (Lombi et al., 2001). Thus differences between the pH of rhizosphere soil and bulk soil can cause processes of adsorption or desorption and precipitation or solubilisation of metals (Lombi et al., 2001). Redox potential can also change pH in the rhizosphere as a consequence of the release of reducing agents by microbial activity. Plant roots release about  175 17% of plant photosynthates into the soil with resulting enhancement of microbial populations and activity (Patra et al., 2006).  8.3.6.2 Metal Fractionation in RS and BS  The metal fractionation in RS and BS is given in Figures 8.6. In general, exchangeable and organic fractions were higher in RS compared to in BS, whereas, oxide and residual fractions were higher in BS than in RS. The organic fraction dominates the rhizosphere soil in the case of Cu, whereas oxide fraction dominates the RS for the other three metals, ie. Pb, Mn and Zn. Residual fraction dominates the BS in the case of all the four studied metals. Even though exchangeable fraction is higher in RS than BS, in certain cases such as Festuca growing soils for Cu, Lolium growing soils for Mn and Combination growing soils for Pb and Zn, the exchangeable metal fraction was higher in BS when compared to RS.  The dominance of the exchangeable fraction in BS when compared to that in RS may be attributed to the sequestering action of root exudates of the corresponding plants resulting in re- distribution of mobile fractions to immobile forms. The rhizosphere is a very dynamic environment governed by the reaction between its various components: soil, plant and micro organisms. This difference in metal fractionation can be attributed to the rhizosphere effect which accounts for the increased microbial biomass and activity occurring in the immediate vicinity of roots. This phenomenon is largely due to the high flux of carbon originating from root exudation (Lombi et al., 2001).            176 Figure 8.6. Metal fractions in Rhizosphere soil and Bulk soil. (a) Cu, (b) Pb, (c) Mn, (d) Zn. RS – Rhizosphere soil, BS – Bulk soil. T0 – without amendments, T1 – with amendments. Mean values, n = 3. F significant at P<0.05. SE (Standard Error) of means given in Appendix E.                                      177  0% 20% 40% 60% 80% 100% RS BS RS BS RS BS RS BS RS BS RS BS RS BS RS BS T0 T1 T0 T1 T0 T1 T0 T1 Lolium Festuca Poa Combination Cu  fra ct io n s (% ) 0% 20% 40% 60% 80% 100% RS BS RS BS RS BS RS BS RS BS RS BS RS BS RS BS T0 T1 T0 T1 T0 T1 T0 T1 Lolium Festuca Poa Combination  Pb  fra ct io n s (% ) 0% 20% 40% 60% 80% 100% RS BS RS BS RS BS RS BS RS BS RS BS RS BS RS BS T0 T1 T0 T1 T0 T1 T0 T1 Lolium Festuca Poa Combination M n  fra ct io n s (% ) 0% 20% 40% 60% 80% 100% RS BS RS BS RS BS RS BS RS BS RS BS RS BS RS BS T0 T1 T0 T1 T0 T1 T0 T1 Lolium Festuca Poa Combination Zn  fra ct io n s (% ) Exch Carbonate Oxide Organic Residual (a) (b) (c) (d)   178 Root exudates include a wide spectrum of organic substances which directly affect the chemical properties of the rhizosphere and in particular the speciation and bio-availability of metals. In addition to root exudates, plant roots themselves can alter the various soil processes which have a major influence on the metal dynamics in the soil (Marschner, 1995).  8.3.6.3. Total metal concentrations in RS and BS  0 20 40 60 80 100 120 140 160 RS BS RS BS RS BS RS BS Cu Mn Pb Zn To ta l m e ta ls  (m g/ kg ) Lolium Festuca Poa Combination  Figure 8.7. Total metal concentrations in Bulk soil and Rhizosphere soil. RS – Rhizosphere soil, BS – Bulk soil. Mean values, n = 3. F significant at P<0.05 for all the four metals. SE of means: Cu – 4.67, Mn – 14.04, Pb – 4.13, Zn – 11.8.  Variations were observed between the total metal concentrations in the rhizosphere soil and bulk soil (Figure 8.7). For Cu and Zn, the concentration in RS of Festuca growing soil was less than that in BS, whereas for Mn, the concentration in RS of Poa growing soil was lower than in BS. In the case of Pb, concentration in RS was lower than that in BS for all plant species. The metal concentration in RS of a particular plant species is governed by the demand of the metal by the plant species. If the demand is high, concentration will be low in RS compared to in BS (Gobran et al., 2004). The potential ligands in the rhizosphere of each plant species are selective for particular metals, which finally controls the metal speciation and bio-availability (Jacynthe, 2007).   179 Root-soil interactions can strongly influence the soil solution chemistry in the rhizosphere (Ksouri et al., 2007). Understanding the rhizosphere influence on the solubility and mobility of metals helps in improving soil remediation techniques such as phytostabilisation. When the plants undergo stress conditions such as deficiencies of nutrients or toxicities of contaminants, biochemical pathways are initiated which cause plant roots to respond by secreting chemicals from the roots to the soil (Salisbury and Ross, 1992). Root exudates may include protons, HCO3- ions, organic acids such as formic acid, acetic acid, citric acid, malic acid etc. These secretions tend to increase nutrient availability and decrease availability of toxicants (Salisbury and Ross, 1992). The ways in which plant roots alter the local chemistry in the rhizosphere controls the metal mobilisation or immobilisation in the soil (Marschner et al., 2007). This explains the differences in the characteristics of rhizosphere soil (RS) and bulk soil (BS), brought out by changes in soil pH, exudation of enzymes and low molecular weight organic molecules, bulk density and water content (Gregory and Hinsinger, 1999). Ten to forty % of the total net C assimilated by plants is released in the form of soluble root exudates and also insoluble materials such as cell walls and mucilage (Marschner, 1998). The release of root exudates provides a conducive microclimate in the rhizosphere by triggering the soil biology and increasing the metal flux into that region. Increased sorption of metals onto bacteria, fungi, organic matter, and plant roots in the rhizosphere leads to retention of metal-pollutants and reduce the exposure to the environment, thereby reducing the existing and associated risks due to metal contamination. However the sustainability of this technique requires long term experiments on phytostabilisation, is a suggested future line of work. .  180 8.4 Conclusions  The important conclusions drawn from the study are as follows –  • An increase in soil pH and a decrease in electrical conductivity (EC) in plots were observed from summer to winter. • Application of soil amendments decreased the exchangeable fraction and plant uptake of all the four metals, Cu, Mn, Pb and Zn. Exchangeable Cu, Pb and Zn were highest in summer while exchangeable Mn was highest in autumn. • Lowest mobile fractions (exchangeable. and carbonate bound) were observed in soils growing Festuca for Cu, Lolium for Mn, and the combination (Lolium, Poa and Festuca) for Pb and Zn. • During summer and autumn, major partitioning was in the organic and oxide fractions for Cu and Pb, while it was in the oxide and exchangeable fractions for Mn and Zn. During winter, it was in the oxide and residual fractions for all the four metals. • Shoot concentrations were highest in summer for Cu and Zn and highest in autumn for Pb and Mn. Root concentrations were highest in winter for all the four metals. • Lowest metal concentrations were observed in Festuca for Cu, Lolium for Mn and the combination (Lolium + Poa + Festuca) for Pb and Zn, with addition of soil amendments. • Soil pH was higher and electrical conductivity lower in the bulk soil when compared to the rhizosphere soil. • Partitioning of metals was mainly in the oxide and residual fractions in the bulk soil, whereas it was in the exchangeable and organic fractions in the rhizosphere soil.  The results clearly demonstrate the effectiveness of growing Lolium perenne L, Festuca rubra L and Poa pratensis L with soil amendments (lime + phosphate) for the phytostabilisation of metal-contaminants (Cu, Pb, Mn and Zn) in the highway soils of southwest British Columbia.  One critical aspect largely missing and requiring more research is the long-term success of phytostabilisation.     181 8.5 References  Adriano, D. C., Wenzel, W. W., Vangronsveld, J., and Bolan, N. S.(2004). Role of assisted natural remediation in environmental cleanup. Geoderma, 122, 121–142.  Alloway, B. J. (1990). Soil processes and the behavior of metals. In: Alloway B. J. (Ed), Heavy metals in soils). London: Blackie, pp. 38–57.  Almas, A., Singh, B. R. and Salbu, B. (1999). Mobility of cadmium-109 and zinc-65 in soil influenced by equilibration time, temperature, and organic matter. J. Environ. Qual., 28, 1742–1750.  Arines, J., Porto, M. E. and Vilarino, A. (1992) Effect of manganese on vesicular-arbuscular mycorrhizal development in red clover plants and on soil Mn-oxidizing bacteria. Mycorrhiza, 1, 127-131.  Basta, N. T. and Tabatabai, M. A. (1992). Effect of cropping systems on adsorption of metals by soils: II. Effect of pH. Soil Sci., 153, 195-204.  Berggren, D. (1989). Speciation of aluminium, cadmium, copper, and lead in humic soil solutions – a comparison of the ion exchange column procedure and equilibrium dialysis. International Journal of Environmental and Analytical Chemistry, 35, 1-15.  Brady, N. C. and Weil, R. R. (1996). The Nature and Properties of Soil (11th edition). Prentice Hall, New Jersey.  Brekken, A. and Steinnes, E. (2004). Seasonal concentrations of cadmium and zinc in native pasture plants: consequences for grazing animals. Science of The Total Environment, 326, 181–195.  Brezonik, P. L., Mach, C. E. and Sampson, C. J. (2003). Geochemical controls for Al, Fe, Mn, Cd, Cu, Pb, and Zn during experimental acidification and recovery of Little Rock Lake, WI, USA. Biogeochemistry, 62, 119–143.  Brooks, R. R. (ed) (1998). Plants that hyperaccumulate heavy metals. Wallingford: CAB International, p. 384.  Caçador, I., Vale, C. and Catarino, F. (2000). Seasonal variation of Zn, Pb, Cu and Cd concentrations in the root-sediment system of Spartina maritima and Halimone portulacoides from Tagus estuary salt marshes. Mar. Environ. Res., 49, 279–290.  Chaney, R. L., Malik, M., Li, Y. M., Brown, S. L., Brewer, E. P., Angle, J. S and Baker, A. J. M. (1997). Phytoremediation of soil metals. Curr. Opin. Biotechnol., 8, 279–284.  Chopin, E. I. B., Marin, B., Mkoungafoko, R., Rigaux, A., Hopgood, M. J., Delannoy, E., Cancès, B. and Laurain, M. (2008). Factors affecting distribution and mobility of trace elements (Cu, Pb, Zn) in a perennial grapevine (Vitis vinifera L.) in the Champagne region of France. Environmental Pollution, 156 (3), 1092-1098.  182  Conesa, H. M., Faz, Á. and Arnaldos, R. (2006). Heavy metal accumulation and tolerance in plants from mine tailings of the semiarid Cartagena-La Unión mining district (SE Spain). Science of The Total Environment, 366, 1–11.  Cox, W. J. and Rains, D. W. (1972) Effect of lime on lead uptake by five plant species. J. Environ. Qual., 1, 167–169.  Djingova, R. and. Kuleff, I. (1994). On the sampling of vascular plants for monitoring of heavy metal pollution. In: B. Markert, Editor, Environmental Sampling for Trace Analysis, VCH, Weinheim, New York, Basel, Cambridge, pp. 395–414.  Doran, J. W. and. Safley, M. (1997). Defining and assessing soil health and sustainable productivity. In: C.E. Pankhurst, B.M. Doube and V.V.S.R. Gupta, Editors, Biological Indicators of Soil Health, CAB International, Wallingford, UK, pp. 1–28.  Duman, F., Olcay, O. and Demirezen, D. (2006). Seasonal changes of metal acumulation and distribution in shining pondweed (Potamogeton lucens). Chemosphere, 65 (11), 2145- 2151.  FHWA. (1998). Is Highway Runoff a Serious Problem? Office of Infrastructure R&D. Turner- Fairbank Highway Research Center. McLean, VA. http:/www.fhwa.dot.gov/terp/prog.htm#I129  Geebelen, W., Vangronsveld, J., Adriano, D. C., Van Poucke, L. C., Clijsters, H. (2002). Effects of Pb-EDTA and EDTA on oxidative stress reactions and mineral uptake in Phaseolus vulgaris. Physiol Plant., 115, 377-384.  Gobran, R. G., Wenzel, W. W. and Lombi, E. (2001). Trace Elements in the Rhizosphere, CRC Press, Washington DC, pp. 321.  Gregory, P. J. and Hinsinger, P. (1999). New approaches to studying chemical and physical changes in the rhizosphere: an overview. Plant and Soil, 211, 1-9.  Hall K. J., Kiffney P., Macdonald R., McCallum D., Larkin G., Richardson J., Schreirer H., Smith J., Zandbergen P., Keen P., Belzer W., Brewer R., Sekela M., Thomson B., (1998). Non-Point Source Contamination in the Urban Environment of Greater Vancouver. A Case Study of the Brunette River Watershed. The Fraser River Action Plan Publications. Environment Canada. Environmental Conservation Branch. Aquatic and Atmospheric Sciences Division, Vancouver.  Hathhorn, W. E. and Yonge, D. R. (1996). The Assessment of Groundwater Pollution Potential Resulting from Stormwater. Infiltration Best Management Research Report, U.S. FHWA.  Hodes, G., Thomas, V. and Williams, A. (2003). A Strategy to Phase-Out Lead in African Gasoline. Renewable Energy for Development, Stockholm Environment Institute, 16(3), 2003.   183 Jacynthe, D. R. (2007). Influence of root activity on speciation and solubility of nutrients and metals in the rhizosphere. Ph.D., Eidgenoessische Technische Hochschule Zuerich (Switzerland).  Jennings, A. A. and Petersen, E. J. (2006). Variability of North American regulatory guidance for heavy metal contamination of residential soil, Journal of Environmental Engineering and Science, 5, 485–506.  Kabata-Pendias, A. and Pendias, H., (2001). In: Trace Elements in Soils and Plants (3rd ed.), CRC Press, Boca Raton, FL.  Kim, N. D. and Fergusson, J. E. (1994). Seasonal variations in the concentrations of cadmium, copper, lead and zinc in leaves of the horse chestnut (Aesculus hippocastaneum L). Environmental Pollution, 86, 89–97.  Ksouri, R., Debez, A., Mahmoudi, H., Ouerghi, Z., Gharsalli, M. and Lachaal, M. (2007). Genotypic variability within Tnusian grapevine varieties (Vitis vinifera L.) facing bicarbonate induced iron deficiency. Plant Physiology and Biochemistry, 45, 315-322.  Kumar, P. B. A. N., Dushenkov, V., Motto, H. and Raskin, I. (1995). Phytoextraction: The use of plants to remove heavymetals from soils. Environ. Sci. Technol., 29(5), 1232-1238.  Kumpiene, J., Lagerkvist, A. and Maurice, C. (2007). Stabilization of Pb- and Cu-contaminated soil using coal fly ash and peat. Environmental Pollution, 145, 365-373.  Lee, S. B., Kwon, S., Park, S., Jeong, M., Han, S., Byun, M. and Daniell, H. (2003). Accumulation of trehalose within transgenic chloroplasts confers drought tolerance. Mol Breed., 11, 1–13.  Lombi, E., Zhao, F. J., McGrath, S. P., Young, S. and Sacchi, A. (2001).Physiological evidence for a high-affinity cadmium transporter highly expressed in a Thlaspi caerulescens ecotype. New Phytol., 149, 53–60.  Ma, J. and. Jennings, A. A. (2008). A model to evaluate internal acid neutralization resistance to soil extraction. Environmental Modelling & Software, 23 (5), 663-669.  Martin, M. H. and Coughtrey, P. J. (1982). Biological Monitoring of Heavy Metal Pollution, Land and Air. Applied Science, London, UK.  Marschner, H. (1995). Mineral nutrition of higher plants. 2nd ed., Academic Press, London.  Marschner, H (1988) Mechanisms of manganese acquisition by roots from soils. In: Graham RD, Hannam RJ, Uren NC (eds) Manganese in soils and plants. Kluwer, London, pp. 191-204.  Marschner, P., Solaiman, Z. and Rengel, Z. (2007). Brassica genotypes differ in growth, phosphorus uptake and rhizosphere properties under P-limiting conditions. Soil Biology and Biochemistry, 39, 87-98.   184 Mattina, M. J., Lannucci-Berger, W., Musante, C. and White, J. C. (2003). Concurrent plant uptake of heavy metals and persistent organic pollutants from soil. Environ. Pollut., 124, 375–378.  McBride, M. B. (1994). Environmental Chemistry of Soils. Oxford University Press, New York.  McGowen, S. L., Basta, N. T. and Brown, G. O. (2001). Use of diammonium phosphate to reduce heavy metal solubility and transport in smelter-contaminated soil. J. Environ. Qual., 30, 493–500.  Mench, M., Vangronsveld, J., Clijsters, H., Lepp, N. W. and Edwards, R. (2000). In situ metal immobilization and phytostabilization of contaminated soils. In: Terry N., Bañuelos G. (eds.): Phytoremediation of Contaminated Soil and Water. Lewis Publ., Boca Raton, London, New York, Washington D.C, pp. 323–358.  Padmavathiamma, P. K. and Li, L. Y. (2007). Phytoremediation Technology: Hyper- accumulation metals in plants. Water, Air, and Soil Pollution, 184, 105-126.  Padmavathiamma, P. K. and Li, L. Y. (2008). Sustainable remediation of Pb for Highway soils. International Conference on Waste Engineering and Management, CSCE-HKIE, Hong Kong, May, 2008.  Padmavathiamma, P. K. and Li, L. Y. (2009a). Phytoremediation of metal-contaminated soil in temperate humid regions of British Columbia, Canada. International Journal of Phytoremediation, 11(6), 575-590.  Padmavathiamma, P. K. and Li, L. Y. (2009b). Phytoremediation and its effect on mobility of metals in soil: a fractionation study. Land Contamination and Reclamation, 17(2), 223-236.  Patra, A. K., Abbadie, L., Clays-Josserand, A., Degrange, V., Grayston, S. J., Guillaumaud, N., Loiseau, P., Louault, F., Mahmood, S., Nazaret, S., Phillippot, L., Poly, F., Prosser, J. I. and Le Roux, X. (2006). Effects of management regime and plant species on the enzyme activity and genetic structure of N-fixing denitrifying and nitrifying bacterial communities in grassland soils. Environmental Microbiology, 8, 1005-1016.  Petrangeli, P. M., Majone, M. and Rolle, E. (2001). Kaolinite sorption of Cd, Ni and Cu from landfill leachates: influence of leachate composition. Water Sci Technol., 44, 343–350.  Preciado, H. F. and Li, L. Y. (2006). Evaluation of metal loadings and bioavailability in air, water and soil along two Highways of British Columbia, Canada. Water, Air, and Soil Pollution, 172, 81–108.  Ross, S. M. (1994). Toxic metals: fate and distribution in contaminated ecosystems. In: Ross, S.M., Editor, 1994. Toxic metals in soil–plant systems. Wiley, Chichester, pp. 189–243.  de la Rosa, G., Peralta-Videa, J. R. and. Gardea-Torresdey, J. L. (2003). Utilization of ICP/OES for the determination of trace metal binding to different humic fractions. Journal of Hazardous Materials, 97, 207–218.  185 SAS. (2001). SAS user's guide: statistics. Cary, NC: SAS Institute. 2001. Sanders, J. R., Adams, T. M. and Christensen, B. T. (1986). Extractability and bioavailability of zinc, nickel, cadmium and copper in three Danish soils sampled 5 years after application of sewage sludge. J. Sci. Food Agric., 37, 1155–1164.  Salisbury, F. B. and Ross, C. W. (1992). Plant physiology. Wadsworth Publishing Co, Belmont, CA  Shuman, L. M. (1999). Organic waste amendments effect on zinc fractions of two soils. J. Environ. Qual., 28, 1442–1447.  Simon L. (2005): Stabilization of metals in acidic mine spoil with amendments and red fescue (Festuca rubra L.) growth. Environ. Geochem. Health, 27, 289–300.  Smith, R. A. H. and Bradshaw, A. D. (1979). The use of metal tolerant plant populations for the reclamation of metalliferous wastes. Journal of Applied Ecology, 16, 595–612.  Sparks, D. L. (2003). Environmental Soil Chemistry, second edition. Academic Press, San Diego, California, p. 368.  Sreevastava, P. C. and Gupta, U. C. (1996). Trace elements in crop production. Science Publishers, Inc., Lebanon, USA.  Strobel, B. W., Hansen, H. C. B., Borggaard, O. K., Andersen, M. K. and Raulund-Rasmussen, K. (2001). Composition and reactivity of DOC in forest floor soil solutions in relation to tree species and soil type. Biogeochemistry, 56, 1–26.  Tessier, A., Cambell, P. G. C. and Bisson, M. (1979). Sequential extraction procedures for the speciation of particulate trace metals. Anal. Chim., 51, 844–851.  Thomson, N. R., Mcbean, E. A., Snodgrass, W. and Manstrenko, B. (1997).  Highway stormwater runoff quality: development of surrogate parameter relationships. Water Air Soil Pollut., 94, 307–347.  Weis, J. S. and Weis, P. (2004). Metal uptake and transport and release by wetland plants: Implication for phytoremediation and restoration. Environmental International, 30, 685– 700.  Wilkins, D. A. (1978). The measurement of tolerance to edaphic factors by means of root growth. New Phytol., 80, 623–633.  186 9. CONCLUSIONS AND RECOMMENDATIONS  9.1 Conclusions  The effect of metal-contaminants on ecosystems and human health is a function of their solubility and mobility (Gobran et al., 2001; Robinson et al., 2005, 2009). These effects are often disproportionate to the metal’s total concentration (Mench et al., 2000). The speciation of metals and the physico-chemical properties of the soil are significant in determining bio-availability. Soil characteristics (eg. pH, clay, organic matter content and type, moisture content etc.) control the speciation of metals, temporary binding by particle surfaces (adsorption-desorption processes), precipitation reactions and availability in soil solution (Adriano et al., 2004). This research project was conducted to develop an effective and environmentally friendly technology to effectively limit the dispersal of metal contaminants to the surrounding natural environment, while maintaining the biological and functional integrity of the soil after remediation.  The preliminary studies (Chapter 3) discussed field measurements of trace element accumulation (Cu, Pb, Mn and Zn) in soils and plants along the highway sites and investigated the phytoremediation potential of plants that spontaneously colonised the site. Concentrations of Cu, Pb, Mn and Zn decreased with increasing distance from the highway. Festuca rubra, which invaded and colonized the contaminated site, was found to be an ideal candidate for the subsequent phytostabilisation studies. The moss collected from the study site, Rhytidiadelphus squarrosus, was found to accumulate significant amounts of Pb. Information from this study provided insight to formulate the technical program for the subsequent studies.  The next study (Chapters 4 and 5) evaluated the phytoextraction/phytostabilisation potential of five plant species, Brassica napus L (rape), Helianthus annuus L. (sunflower), Lolium perenne L (perennial rye grass), Poa pratensis L (Kentucky blue grass) and Festuca rubra L (creeping red fescue) for metals (Cu, Pb, Mn and Zn) in soils with different metal contamination levels. Soil metal (Cu, Pb, Mn and Zn) fractionation and metal accumulation characteristics in plants were studied at two stages of plant growth, 90 and 120 DAS (days after sowing). In soils with total Cu, Pb, Mn, and Zn concentrations of 520, 1100, 2160, and 1600 mg/kg respectively, none of the seeds germinated, maybe because of metal toxicities (Adriano, 2001).   187 In the study investigating the accumulation characteristics and translocation properties of metals (Cu, Pb, Mn and Zn) in different plant species (Brassica napus, Festuca rubra, Helianthus annuus, Lolium perenne, and Poa pratensis ) (Chapter 4), Enrichment Coefficients (EC) of root and shoot and Translocation Factors (TF) were discussed in addition to metal absorption and metal uptake values. The metal concentrations in plants increased by nearly a factor of two for Cu and Pb, three times for Mn and four times for Zn in BA soils, compared to B0  soils . The efficiency of plants to accumulate metals followed the order, Festuca > Lolium > Helianthus > Poa > Brassica for Cu, Helianthus > Brassica > Festuca > Poa > Lolium for Pb, Poa > Festuca > Lolium > Brassica > Helianthus for Mn and Helianthus > Festuca > Poa > Brassica > Lolium for Zn. Metal removal was higher at 120 DAS than at 90 DAS, and metals concentrated more in below-ground tissues with less translocation to the above-ground parts. EC (Enrichment Coefficient) values indicate that Festuca had the highest accumulation for Cu, Helianthus for Pb and Zn, and Poa for Mn. Metal uptake values were lowest for Festuca and highest for Lolium among the plant species studied, demonstrating that metal content or uptake is more important than metal concentration in a phytoremediation study.  The effect of plant growth on the re-distribution of metal fractions (Chapter 5) at two different stages of plant growth (90 and 120 DAS) showed that there was a decrease in the exchangeable fraction and an increase in the oxide and organic fractions of metals in soils. The oxide fraction of metals dominated in Festuca soils, organic fraction in Lolium and Poa soils and exchangeable fraction in Helianthus and Brassica soils. There was a significant partitioning of metal fractions to insoluble forms by the growth of Festuca for Cu, Poa for Pb and Zn and Lolium for Mn. The relative partitioning of metals shifted more to the immobile forms as the growth of plants advanced from 90 to 120 DAS. The percentage migration by leaching from the soil was more for Cu and Pb than for Mn and Zn. Based on the results, Lolium, Poa and Festuca were identified to be suitable for phytostabilisation of Cu, Pb, Mn and Zn in moderately contaminated acid soils.  Chapter 6 outlines the effect of soil amendments in modifying soil properties and thereby influencing the plants to immobilise Cu and Zn. Plant species Lolium perenne, Festuca rubra and Poa pratensis were tested in the presence of soil amendments (lime, phosphate and compost, both individually and in combination) to assess the effect of soil-plant-amendment interaction on phytostabilisation of Cu and Zn. Changes in soil pH due to the application of lime had a significant effect on the exchangeable fractions of Cu and Zn, organic fractions of Cu and oxide  188 fractions of Zn in soil. Maximum metal immobilisation was achieved in the soil by the combined application of amendments (lime + phosphate + compost), in conjunction with growth of Festuca for Cu and Poa for Zn. Application of lime significantly reduced the exchangeable fraction of Cu and Zn. Phosphate application increased the exchangeable Cu content of soil and enhanced plant Cu, whereas it led to decreased plant Zn. Lowest EC and TF values were observed in Festuca for Cu and Poa for Zn, with the combined application of amendments. Zn exhibited the highest mobility, as shown by exchangeable Zn fraction in the soil. It is likely that its accumulation will remain in the highway environment for years to come due to persistent sources of this metal, e.g. tire rubber, motor oil, grease, etc. (Viklander, 1998; Varrica et al., 2003).  In the study investigating the effect of plant growth and amendment addition on Pb and Mn immobilisation (Chapter 7), addition of soil amendments to accelerate physico-chemically driven sorption processes and growth of appropriate plant species to reduce physiologically driven uptake of Pb and Mn were investigated. Lolium perenne, Festuca rubra and Poa pratensis were tested in the presence of soil amendments (lime, phosphate and compost, both individually and in combination). Lime application lowered plant Pb and Mn concentrations, while phosphate application decreased the plant Pb and increased the plant Mn. Addition of lime re-distributed >40% of total Pb to the oxide fraction whereas addition of phosphate re-distributed >62% of total Pb to the residual fraction. Phosphate addition increased the exchangeable Mn fraction by 35% and the combined application of amendments lowered the exchangeable Mn fraction by almost 50%. Combined amendment addition resulted in a significant decrease in the exchangeable (mobile) metal fraction in soils growing Poa for Pb and in soils growing Lolium for Mn. ECroot and ECshoot for Pb in Poa decreased by 72 and 60% with the combined application of amendments, while the corresponding decreases for Mn in Lolium were 48 and 43%.  Chapter 8 reports on the field experiment conducted at the study site to confirm the results from the pot experiments. The primary objectives of this study were: (1) to quantify the seasonal effect of metal accumulation in soil and to assess the seasonal impact on the metal speciation in the soil by the influence of soil amendments and different plant species; (2) to determine accumulation differences between sampling periods in plant parts and to identify the plant part accumulating significantly higher amounts of metals seasonally; and (3) to assess the influence  189 of root-soil interactions on metal dynamics. The final outcome of the study was the development of a remediation strategy for metals (Cu, Pb, Mn and Zn) involving suitable plants and amendments incorporating seasonal and rhizosphere influences and maintaining the functional and biological integrity of soil after remediation. Application of amendments decreased the exchangeable fraction and plant uptake of all four metals, Cu, Mn, Pb and Zn. Exchangeable Cu, Pb and Zn were highest in summer and exchangeable Mn, highest in autumn. Lowest mobile fractions (exchangeable. and carbonate bound) were observed in soils growing Festuca for Cu, Lolium for Mn, and a combination (Lolium, Poa and Festuca) for Pb and Zn. During summer and autumn, Cu and Pb were mainly partitioned in the organic and oxide fractions, whereas Mn and Zn were partitioned in the oxide and exchangeable fractions. During winter, major partitioning was in the oxide and residual fractions for all four metals. Lowest metal entry into plants was observed to occur as follows: Festuca for Cu, Lolium for Mn and the combination (Lolium + Poa + Festuca) for Pb and Zn. The soil pH was higher and electrical conductivity lower in bulk soil compared to rhizosphere soil. Partitioning of metals was mainly into the oxide and residual fractions in the bulk soil, whereas it was in the exchangeable and organic fractions in the rhizosphere soil.  The studies conducted on the remediation potential of plants in soils from two highway sites with similar geometric designs, but different conditions (i.e. climate, land use, daily traffic, geology, etc.) showed common patterns of metal fractionation in soil and metal accumulation characteristics in plants. Among the plants studied, Lolium perenne L, Festuca rubra L and Poa pratensis L are promising with respect to immobilising metal contaminants in soil and can be recommended for practical phytostabilisation. However this dissertation did not collect data related to the cost-effectiveness of phytostabilization to limit the dispersal of metal contaminants in highway soils.  9.2. Recommendations and future work  Lolium perenne L, Festuca rubra L and Poa pratensis L have been identified as being suitable for phytostabilisation of metal-contaminants (Cu, Pb, Mn and Zn) in highway soils. Significant partitioning of metal fractions to insoluble forms was achieved by the growth of Festuca for Cu, Poa  190 for Pb and Zn and Lolium for Mn. The speciation of metals and the physico-chemical properties of the soil are significant in determining bio-availability and mobility of metals in soils rather than the total metal concentration in the soil. Addition of soil amendments such as lime, phosphate and compost complement the plant effect in metal immobilisation by maintaining a favourable soil pH, and increasing the metal sorption in soil. Growing these plant species along highway soils could help to reduce the metal concentration of highway runoff by filtering or trapping metal-containing particulates and reducing the amount of metal-contaminated sediments entering the biota. Since the distribution and association of metals with various soil fractions directly affect mobility and bioavailability (Robinson et al., 2005), continuous monitoring of metal partitioning during different plant growth stages is essential to prevent associated risks. The mobile-immobile distributions of metal fractions, controlled by various pedogenic and biogenic processes, are influenced by seasonal changes (Duman et al., 2006; Kim and Fergusson, 1994). Hence monitoring the seasonal influence on metal fractionation would help to forecast the environmental pollution from metal contaminants in the soil.  Even though there is variability with respect to plant species in re-distributing metal fractions in soil (Robinson et al., 2009), considering the metal fractions of environmental importance, the proportion of metals bonded to oxides was higher than the proportion associated with organic and residual fractions. Reasons for this observation require further elaboration.  The effects of combined metals on plant metal uptake are complex (Ebbs and Kochian, 1997). Further studies are required to elucidate the interactions (antagonism or synergism) between metals at different concentration levels.  No attempt was made in the current work to assess the presence and concentration of the appropriate transport proteins or translocating chelating molecules in plants. It is known that these compounds play a very important role in the translocation of metals (Hiromura and Sakurai, 2001), and this should be investigated in the future studies.  The metal speciation due to aging in the spiked soil and the change in microbial biomass and mycorrhization are also suggested as subjects for future studies.   191 Mechanisms may vary among different plant species to cope with metal exposure. Hence studies on metal detoxification in the plant species tested may yield a better understanding of molecular mechanisms for metal-tolerance, an important topic for future work.  Identified plants should be investigated for their physiological aspects, especially stress physiology and exposure to sUVB (supplemental UVB) radiation under different levels of metal- contaminations.  Characterisation of root exudates of different plant species to identify metal specific organic ligands may help to explain the differential responses of plants to metal-partitioning in soil.  Impact of metal sequestration in roots and in the rhizosphere on soil micro and macro fauna and whether this will cause off-site concerns need further study.  Characterization of soil in identifying its mineral or material oxide and residual fractions of metals may provide a better understanding of the fate of metal-contaminants in soil.  One critical aspect largely missing at this point is the long-term success of phytostabilisation. Hence long-term studies are recommended to help evaluate the efficacy of phytostabilisation in remediating metal toxicity, in promoting plant succession, and in maintaining the functional integrity of soil after remediation.                   192  9.3. References  Adriano, D. C. (2001). Trace Elements in Terrestrial Environments: Biogeochemistry, Bioavailability and Risks of Metals (Second edition), Springer-Verlag, New York.  Adriano, D. C., Wenzel, W. W., Vangronsveld, J. and Bolan, N. S.(2004). Role of assisted natural remediation in environmental cleanup. Geoderma, 122, 121–142.  Duman, F., Olcay, O. and Demirezen, D. (2006). Seasonal changes of metal acumulation and distribution in shining pondweed (Potamogeton lucens). Chemosphere, 65 (11), 2145- 2151.  Ebbs, S. D and. Kochian, L. V (1997). Toxicity of zinc and copper to Brassica species: implications for phytoremediation. J. Environ. Qual., 26, 776–781.  Gobran, R. G., Wenzel, W. W. and Lombi, E. (2001). Trace Elements in the Rhizosphere. CRC Press, Washington DC, pp. 321.  Hiromura, M. and Sakurai, H. (2001). Intracellular metal transport proteins, RIKEN Review No. 35, 23-25.  Kim, N. D. and Fergusson, J. E. (1994). Seasonal variations in the concentrations of cadmium, copper, lead and zinc in leaves of the horse chestnut (Aesculus hippocastaneum L). Environmental Pollution, 86, 89–97.  Mench, M., Vangronsveld, J., Clijsters, H., Lepp, N. W and Edwards, R. (2000): In situ metal immobilization and phytostabilization of contaminated soils. In: Terry N., Bañuelos G. (eds.): Phytoremediation of Contaminated Soil and Water. Lewis Publ., Boca Raton, London, New York, Washington D.C, pp. 323–358.  Robinson, B. H., Bolan, N. S., Mahimairaja, S. and Clothier, B. E. (2005). Solubility, mobility and bioaccumulation of trace elements: abiotic processes in the rhizosphere. In: Trace Elements in the Environment: Biogeochemistry, Biotechnology, and Bioremediation (Eds. MNV Prasad, KS Sajwan, R Naidu). CRC press, Boca Raton Florida, pp. 97 – 110. Robinson, B. H., Bañuelos, G. S., Conesa, H. M., Evangelou, M. W. H. and Schulin, R. (2009). The phytomanagement of trace elements in soil. Critical Reviews in Plant Sciences, 28(4), 240-266. Varrica, D., Dongarra, G., Sabatino, G. and Monna, F. (2003). Inorganic Geochemistry of Roadway Dust from the Metropolitan Area of Palermo, Italy. Environmental Geology, 44, 222-230.  Viklander, M. (1998). Particle size distribution and metal content in street sediments. Journal of Environmental Engineering, 124, 761-766.   193  APPENDICES   APPENDIX A - PRELIMINARY STUDIES                         - Main Site Background site  Figure A.1 Satellite Photograph (HW1 - Study site)                           Main Site Background site  Figure A.2 Satellite Photograph (HW17 - Study site)     194  Map of Canada           Figure A.3 Location of study site (HW 1)                           Location of study site (HW 17)  195   Figure A.4 HW 1 study site       Figure A.5 HW 17 study site  196   Figure A.6 Plants collected from the HW1 study site in winter    Figure A.7 Plants collected from the HW1 study site in summer  197                  Lamium purpureum                                                       Plantago lanceolata                       Agrostis exerata                                                        Ranunculus repens               Ranunculus occidentalis                                                       Holcus lanatus   Figure A.8  Plants collected from the HW17 study site in summer  198 Table A.1. Summary of Trans-Canada Highway characteristics in Surrey, B.C and Highway 17 characteristics in Delta, B.C.   Characteristics Trans-Canada Hwy (HW 1) Highway 17 Type Mixed residential, industrial and parkland Rural Average Daily Traffic (ADT) 82,900 vehicles (Westbound- WB) 73,100 vehicles (Eastbound- EB) 20,417 vehicles (Northbound- NB) 22,899 vehicles (Southbound- SB) Drainage area 500 m 2  1286m2  (NB) 1368m2 (SB) Surface pavement Asphalt Asphalt Number of lanes/direction 3 2 Type of section Elevated flush shoulder Elevated flush shoulder Surrounding land use Agricultural, residential Agricultural                                 199 APPENDIX B - STAGE 1 STUDY Stage 1 - Experimental Design 1 2 3 4 5 6 7 8 9 10 11 12 21 22 23 24 25 26 27 28 29 30 31 32 41 42 43 44 45 46 47 48 49 50 51 52 13 14 15 16 17 18 19 20 33 34 35 36 37 38 39 40 53 54 55 56 57 58 59 60 71 72 73 74 75 76 77 78 79 80 61 62 63 64 65 66 67 68 69 70 81 82 83 84 85 86 87 88 89 90 P1T1R1S1 P1T1R2S1 P1T1R1S2 P1T1R2S2 P1T2R1S1 P1T2R2S1 P1T2R1S2 P1T2R2S2 P1T3R1S1 P1T3R2S1 P1T3R1S2 P1T3R2S2 P2T1R1S1 P2T1R2S1 P2T1R1S2 P2T1R2S2 P2T2R1S1 P2T2R2S1 P2T2R1S2 P2T2R2S2 P2T3R1S1 P2T3R2S1 P2T3R1S2 P2T3R2S2 P3T1R2S1 P3T1R2S1 P3T1R1S2 P3T1R2S2 P3T2R2S1 P3T2R2S1 P3T2R1S2 P3T2R2S2 P3T3R2S1 P3T3R2S1 P3T3R1S2 P3T3R2S2 P4T1R1S2 P4T1R2S1 P4T1R1S2 P4T1R2S2 P4T2R1S2 P4T2R2S1 P4T2R1S2 P4T2R2S2 P4T3R1S2 P4T3R2S1 P4T3R1S2 P4T3R2S2 P5T1R2S2 P5T1R2S1 P5T1R1S2 P5T1R2S2 P5T2R2S2 P5T2R2S1 P5T2R1S2 P5T2R2S2 P5T3R2S2 P5T3R2S1 P5T3R1S2 P5T3R2S2 P1T1R3S1 P1T1R3S2 P1T2R3S1 P1T2R3S2 P1T3R3S1 P1T3R3S2 P2T1R3S1 P2T1R3S2 P2T2R3S1 P2T2R3S2 P2T3R3S1 P2T2R3S2 P3T1R3S1 P3T1R3S2 P3T2R3S1 P3T2R3S2 P3T3R3S1 P3T3R3S2 P4T1R3S1 P4T1R3S2 P4T2R3S1 P4T2R3S2 P4T3R3S1 P4T3R3S2 P5T1R3S1 P5T1R3S2 P5T2R3S1 P5T2R3S2 P5T3R3S1 P5T3R3S2 Plants P1 – Brassica napus P2 – Helianthus annuus P3 – Lolium perenne P4 – Festuca rubra P5 – Poa pratensis Treatments T1 – Initial   Soil T2 – Soil spiked with A level metals T3 – Soil spiked with C level metals Replications : R1, R2, R3  Stage 1 - Experimental Design   1. P1T1R1S1 2. P1T1R2S1  3. P1T1R1S2 4. P1T1R2S2  5. P2T1R1S1  6.  P2T1R2S1  7.  P2T1R1S2  8.  P2T1R2S2  9. P3T1R2S1  10.  P3T1R2S1  11.  P3T1R1S2  12.  P3T1R2S2  13. P4T1R1S2  14.  P4T1R2S1  15.  P4T1R1S2  16.  P4T1R2S2  17. P5T1R2S2  18.  P5T1R2S1  19.   P5T1R1S2  20.  P5T1R2S2   21. P1T2R1S1  22.  P1T2R2S1  23.  P1T2R1S2  24.  P1T2R2S2  25. P2T2R1S1  26.  P2T2R2S1  27.  P2T2R1S2  28.  P2T2R2S2  29. P3T2R2S1  30.  P3T2R2S1  31.  P3T2R1S2  32.  P3T2R2S2  33.  P4T2R1S2  34.  P4T2R2S1  35.  P4T2R1S2  36.  P4T2R2S2  37. P5T2R2S2  38.  P5T2R2S1  39.   P5T2R1S2  40.  P5T2R2S2   41. P1T3R1S1     42.  P1T3R2S1      43.  P1T3R1S2    44.  P1T3R2S2  45. P2T3R1S1  46.  P2T3R2S1      47.  P2T3R1S2    48.  P2T3R2S2  49. P3T3R2S1  50.  P3T3R2S1      51.  P3T3R1S2    52.  P3T3R2S2  53.  P4T3R1S2  54.  P4T3R2S1      55.  P4T3R1S2    56.  P4T3R2S2  57. P5T3R2S2  58.  P5T3R2S1      59.   P5T3R1S2    60.  P5T3R2S2   61. P1T1R3S1   62.  P1T1R3S2         71.  P1T2R3S1  72.  P1T2R3S2 81.  P1T3R3S1  82.  P1T3R3S2  63. P2T1R3S1   64.  P2T1R3S2         73.  P2T2R3S1  74.  P2T2R3S2  83.  P2T3R3S1  84.  P2T2R3S2  65. P3T1R3S1  66.  P3T1R3S2         75.  P3T2R3S1  76.  P3T2R3S2         85.  P3T3R3S1   86.  P3T3R3S2  67. P4T1R3S1  68.  P4T1R3S2         77.  P4T2R3S1  78.  P4T2R3S2          87.  P4T3R3S1  88.  P4T3R3S2  69. P5T1R3S1  70.  P5T1R3S2         79.   P5T2R3S1  80.  P5T2R3S2         89.  P5T3R3S1  90.  P5T3R3S2 Plants P1 – Brassica napus P2 – Helianthus annuus P3 – Lolium perenne P4 – Festuca rubra P5 – Poa pratensis Treatments T1 – Initial   Soil T2 – Soil spiked with A level metals T3 – Soil spiked with C level metals Replications : R1, R2, R3   Figure B.1 5*3*3 = 45 pots. Two sets for destructive sampling at 90 and 120 DAS (45*2 = 90). T1 – B0, T2 – BA, T3 – BC. S1 – 90 DAS, S2 – 120 DAS.   200 Table B.1. Sequential chemical extractions for the partitioning of metals and their respective reagents   Fraction  Metal Species  Extracting Reagent Treatment 1  Exchangeable  1 M KNO3 adjusted to natural soil pH 25 o C 1 hour agitation 2  Carbonate  1 M NaOAc, adjusted to pH 5 with HOAc 25 o C 1 hour agitation 3  Oxide  0.04 M NH2OH⋅HCl in 25% (v/v) HOAc 96 o C, 6 hours with intermittent agitation 4  Organic  30% H2O2 (pH 2 w/ HNO3) + 0.02 M HNO3 1) 85 o C, 2 h intermittent agitation; 2) addition of (H2O2), 3 h intermittent agitation; 3) 3.2 M (NH4OAc) in 20% (v/v) HNO3 continuous agitation for 30 minutes. 5  Residual  HNO3/HCl Complete digestion Reference - Tessier et al (1979) as modified by Preciado and Li (2006)  The solubility of metal fractions is in the order: exchangeable > carbonate specifically adsorbed > Fe-Mn oxide > organic > residual. Water-soluble and exchangeable forms of metals in soils are considered readily mobile and available to plants, whereas metals incorporated into crystalline lattices of clays (residual forms) appear to be relatively inactive. Metals, precipitated as carbonates, occluded in Fe, Mn, and Al oxides, and complexed with organic matter, could be strongly bound in soils, depending on the actual composition, physical and chemical properties of soil.                 201 APPENDIX C - STAGE II STUDY      1. P1T1R1  2.  P1T1R 2  3.  P1T 1R 3  4.  P1T2R 1  5.  P1T2R 2  6.  P1T2R3  7. P1T3R1  8.  P1T3R 2   9.  P1T 3R 3  10.  P1T4R 1  11.  P1T4R 2  12.  P1T4R3  13.  P1T5R1  14.  P1T5R 2  15.  P1T 5R 3  16.  P1T6R 1  17.  P1T6R 2  18.  P1T6R3  19. P2T1R1  20.  P2T1R 2  21.  P2T 1R 3  22.  P2T2R 1  23.  P2T2R 2 24.  P2T2R3  25. P2T3R1  26.  P2T3R 2   27.  P2T 3R 3  28.  P2T4R 1  29.  P2T4R 2  30.  P2T4R3  31.  P2T5R1  32.  P2T5R 2  33.  P2T 5R 3  34.  P2T6R 1  35.  P2T6R 2  36.  P2T6R3  37.   P3T1R1  38.  P3T1R 2  39.  P3T 1R 3  40.  P3T2R 1  41.  P3T2R 2  42.  P3T2R3  43. P3T3R1  44.  P3T3R 2   45.  P3T 3R 3  46.  P3T4R 1  47.  P3T4R 2  48.  P3T4R3  49.  P3T5R1  50.  P3T5R 2  51.  P3T 5R 3  52.  P3T6R 1  53.  P3T6R 2  54.  P3T6R3 Plants - 3 P1 – Lolium P2 – Festuca P3 – Poa Treatm ents  - 6 T1 – Initial soil alone T2 - Initial soil + A level spiking of m etal contam inants (Cu, Pb, M n and Zn) T3 – T2 + Lim e T4 – T2 + P T5 – T2 + Com post T6 – T2 + Lim e + P + Com post Replications : R1, R2, R3 Stage 2 - Experim ental Design   Figure C.1 Layout of the stage II experiment (6*3*3 = 54 pots. Stage of sampling – 90 DAS)   202 Application of amendments  Lime - The recommended dose of lime was 10 tons/ha from the lime requirement estimated (McLean, 1982). Dolomite (finely ground) was used as the liming material. CaCO3 (52.48 %) MgCO3 (40.96 %) Neutralizing value as CaCO3 equivalent (101 %) Screen size – 100 % passing 10 MESH (1.70 mm) 80 % passing 20 MESH (0.85 mm) 30 % passing 100 MESH (0.15 mm).  Phosphate – The recommended dose was 135 kg P2O5/ha, based on the available P status of the soil (10.5 ppm). The source of P used was Ca HPO4. 2H2O (41 % P2O5).  Compost - City of Vancouver Yard Trimming Compost (pH - 6.4; electrical conductivity - 3.2 dSm-1; C/N ratio - 21.3, Cu – 1.2 mg/kg, Zn - 42 mg/kg, Fe – 61 mg/kg and Mn - 146 mg/kg) was used. The recommended dose was 10 tons/ha based on the organic matter status of the soil.  McLean, E. O. (1982). Soil pH and Lime requirement. In: Page, A.L., Miller, R.H. and Keeney, D. R. eds. Methods of Soil Analysis. Madison, Wisconsin USA. p. 214.                           203  Figure C.2 One month after sowing                                          Two months after sowing           Figure C.3 Harvest stage (Three months after sowing)     204     Figure C.4    205  Table C.1. Summary of weather data at UBC from May 2006 to December, 2006, during pot experiments.  Month Mean temperature (°C) Average humidity (%) May, 2006 12.6 81 June, 2006 15.6 84 July, 2006 17.9 83 August, 2006 17.1 87 September, 2006 14.9 89 October, 2006 10.1 93 November, 2006 5.5 96 December, 2006 4.6 96     Table C.2. Root and Shoot biomass, dry weight (DW) (g/pot)  Treatments Root weight (g/pot) Shoot weight (g/pot) Total biomass (g/pot) B0  0.54bc 0.79c 1.33 BA 0.52c 0.71c 1.23 BAL 0.68ab 1.00b 1.68 BAP 0.74a 1.26ab 2 BAO 0.50c 1.4a 1.9 Lolium BALPO 0.77a 1.50a 2.27  B0  0.51c 0.63cd 1.14 BA 0.28d 0.42e 0.7 BAL 0.49c 0.87c 1.36 BAP 0.58b 1.02b 1.6 BAO 0.57bc 1.15b 1.72 Festuca BALPO 0.61bc 1.25ab 1.86  B0  0.63b 0.59d 1.22 BA 0.50 0.52d 1.02 BAL 0.63b 0.68cd 1.31 BAP 0.64b 1.17b 1.81 BAO 0.74a 1.28ab 2.02 Poa BALPO 0.76a 1.32a 2.08  F *  *  *  Mean values, n = 3. * F significant at P<0.05. Significantly different statistical values (P<0.05) according to the Least Significance Test in each column are followed by different letters. B0 – Initial soil, BA – Spiked soil, BAL - Spiked soil plus lime, BAP - Spiked soil plus phosphate, BAO - Spiked soil plus compost, BALPO - Spiked soil plus lime, phosphate and compost.     206  APPENDIX D - STAGE III STUDY    Figure D.1 Experimental Design Stage III        207  50 cm 50 cm v Ist sampling 2nd sampling 3rd sampling    1m Figure D.2 Lay out of each plot  Application of amendments  Lime - The recommended dose of lime was 10 tons/ha from the lime requirement estimated. Dolomite (finely ground) was used as the liming material. Phosphate – The recommended dose was 135 kg P2O5/ha, based on the available P status of the soil. The source of P used was Ca HPO4 2H2O (41 % P2O5).   Table D.1. Summary of weather data at Tsawwassen, Deltaport Way from May 2007 to February, 2008, during the field experiment.   Month Mean temperature (°C) Total precipitation (mm) May, 2007 12.8 37 June, 2007 15.2 80 July, 2007 18.8 53 August, 2007 17.8 8.4 September, 2007 14.2 73.6 October, 2007 9.6 155.2 November, 2007 5.9 116.2 December, 2007 3.2 181.6 January, 2008 5.5 137.6 February, 2008 8.6 68.6 1m  208                        Figure D.3 First sampling (90 days after sowing)    209      Figure D.4 Second sampling (180 days after sowing)      210       Figure D.5 Third sampling (270 days after sowing)     211       APPENDIX E - QA/QC. ABSTRACT OF ANOVA – FIELD EXPERIMENT  E.1. QA/QC  Pot experiments –Stage I and stage II experiments are conducted in CRD (Completely Randomized Design) with three replications. After a thorough mixing of treatments with soil, the moisture content was maintained at field capacity by taking weight of pots and adding the required amount of water. Randomization of pots to ensure uniform distribution of growth conditions was done every week. For stage 1 experiment, there were two stages of sampling, 90 and 120 DAS to find out the metal partitioning and metal removal at two different stages of plant growth. Since Brassica flowered first and the maximum flowering was at 90 DAS and senescence at 120 DAS, all samples were collected at the same time for consistency and comparability. For stage II experiment, there was only one sampling stage since the aim was to evaluate the effect of soil amelioration in influencing the plants to stabilize soil metals.  Field experiment - The design for the field experiment was Completely Randomized Factorial Experiment in Split Plot Design with three replications. Soil and plant samples were collected from all plots at 90, 180 and 270 DAS, corresponding summer, autumn and winter. Each plot was demarcated to three portions with flag stakes for uniform sampling during three different seasons.  E.1.1 Varian Spectre AA 220 Multi-element Fast Sequential Atomic Absorption Spectrometer  QA/QC procedures and protocols consisted of 1 replicate analysis in 20 samples, method blanks, blank spikes and matrix spikes, 1 per batch. Multipoint calibrations were performed daily and verified every 20 samples, with a tolerance of ± 10% of initial calibration. The accuracy of the methodology was evaluated by analyzing two certified reference soil samples (CSSC-1 and CSSC- 2).        212  E.1.2. Standard reference soil samples  Table E.1. Total metal concentrations (mg/kg) (n = 3) Obtained values Soil Cu (mg/kg) Pb (mg/kg) Mn (mg/kg) Zn (mg/kg) CSSC1 48.6 28.4 1398.9 99.6 CSSC2 49.2 17.2 845.4 69.7 Reported values Soil Cu (mg/kg) Pb (mg/kg) Mn (mg/kg) Zn (mg/kg) CSSC1 42 24 1403 91 CSSC2 43 12 874 75 (Mckeague, J. A.,Shedrick, B. H. and Desjardins, J. G. (1978). Compilation of data for CSSC reference soil samples. Soil Research Institute, Ottawa).                                    213 E.1.3. Comparison of ∑SSE with total metal concentrations  Table E.2. Initial values, before plant growth (HW 1)  Treatment Exch. (mg/kg) Oxide (mg/kg) Organic (mg/kg) Residual (mg/kg) Total SSE (mg/kg) Total metal (mg/kg) R.D (%) Cu B0 2.1 11.6 17.6 31 63 52 6 BA  3.7 22.9 28.1 34 89 80 11 Pb B0 3.1 40.9 12.7 28.1 84.8 93 11 BA 9.3 50.9 19.4 34.7 115.3 146 4 Mn B0 10.3 119.7 9.3 78.1 207 223 13 BA 27.8 238.9 23.4 80 370 418 8 Zn B0 9.2 38.7 14.3 20.2 82.4 70 7 BA 34.9 62.8 18.2 54.5 170.4 148 8 n = 3                              214 Table E.3. Stage 1 (90 DAS)  R.D (%) Total concn. (mg/kg) Sum SSE Residual (mg/kg) Organic (mg/kg) Oxide (mg/kg) Exch. (mg/kg) Treatments  Cu 6 45 47.9 15.6 18.6 11.7 2.0 LB0 5 74 77.9 25.8 26.9 23.8 1.3 LBA 3 49 50.1 22.2 15.1 12.3 0.6 FB0 4 78 74.9 26.2 25.3 19.6 1.0 FBA 4 50 48.2 21.1 15.7 10.9 0.5 HB0 2  46 45.3 15.1 18.9 10.6 0.7 PB0 7 75 70.6 23.5 21.5 24.3 1.4 PBA 10 47 42.9 18.1 13.4 10.1 1.3 BrB0 Pb 13 86 98.3 38.3 29.3 27.4 3.3 LB0 9 132 144.3 40 47.5 50.5 6.37 LBA 8  90 89.2 35 22.1 29.7 2.5 FB0 3 140 135.9 48.1 33.4 45.8 4.0 FBA 3 88 85.8 23.3 25 33.1 2.4 HB0 1 89 80.51 27.7 25.6 27.2 1.8 PB0 2 137 134.2 38.7 44.6 42.9 3.9 PBA 9 90 83 26.7 24.6 29.9 1.7 BrB0 Mn 10 207 189 65.0 29.6 89.4 5.1 LB0 4 381 367 119.4 77.4 151.2 19.7 LBA 13  209 186 56.6 29.1 91.7 9.8 FB0 14 396 350 117.8 57 152.1 24.7 FBA 7 210 196 52.9 33.3 97.8 11.4 HB0 8  203 188 48.7 28.0 105.1 7.6 PB0 9 380 351 117.9 50.5 158.7 24.2 PBA 7 211 197 49.8 29.6 105.3 11.4 BrB0 Zn 16 64 76 26 18 23 9 LB0 15 138 120 34 44 46 21 LBA 18  67 80 30 18 25 7 FB0 9 142 156 46 39 48 20 FBA 36 76 56 15 14 19 8 HB0 11  65 73 27 15 25 6 PB0 10 144 132 27 37 47 18 PBA 16 63 75 28 16 23 8 BrB0 n =3        215 Table E.4. Stage II (soils grown with Poa pratensis)  SSE extraction (mg/kg) Treatments Exch. Oxide Organic Residual Total SSE Total concn. (mg/kg) R.D(%) Cu BAL 1.8 25.1 26.0 26.3 79.2 85 7 BAP 5.7 20.6 22.9 15.3 64.5 74 13 BAO 2.0 19.9 29.4 18.3 69.7 78 11 BALPO 0.9 16.5 35.8 23.7 76.9 79 3 Pb BAL 1.10 56.9 17.5 51 126.5 140 10 BAP 0.60 43.8 6.1 72 122.5 134 9 BAO 0.19 30.1 40.5 44 114.79 133 14 BALPO 0.05 27.3 23.9 71 122.25 142 15 Mn BAL 7.6 282 9.4 78 377 386 2 BAP 19.7 231 8.2 52 310.9 331 6 BAO 14.7 216 19.5 59 309.2 307 0.1 BALPO 6.4 284 15.9 51 357.3 390 9 Zn BAL 0.72 74.8 18.5 65.7 160 142 13 BAP 16 38.5 13.1 55.8 123 106 16 BAO 27.1 48.8 23.1 30 119 137 14 BALPO 2.9 74.8 36.1 47 156 139 12 n = 3                        216 Table E.5. Stage III (T0 at 90 DAS)  Plants Exch. (mg/kg) CO3 (mg/kg) Oxide (mg/kg) Organic (mg/kg) Residual (mg/kg) ∑SSE Total concn. (mg/kg) R.D (%) Cu P1 4.7 8.3 5.2 14.5 13.7 46.4 72.7 36 P2 6.1 5.9 11.6 13.7 15 52.3 67.8 23 P3 4 6.8 18.4 17.8 14.4 61.4 81.2 24 P4 6.2 4.7 12.9 17 23.9 64.7 91.9 29 Pb P1 2.9 5.6 18.3 35.2 15.1 77 87.3 12 P2 5.7 8 14.1 19.8 29.3 76.9 81.7 6 P3 0.6 5.5 18.7 44.9 17.6 87.3 93.8 7 P4 3.2 7.8 10.8 22.3 22.9 67 85.8 22 Mn P1 15.4 3.5 45.6 7.9 55.1 127.5 144 11 P2 12.3 5.1 54.7 11 67.7 150.8 126 20 P3 16.3 1.5 59.3 8.9 61.3 147.3 164 10 P4 14.3 3 47 12.8 60.9 138 132 5 Zn P1 23.5 6.9 28.9 48.9 48.5 156.7 182 14 P2 18.9 11.8 38.8 85.7 34.4 189.6 154 23 P3 19.7 18.7 28.6 80.1 42.7 189.8 161 18 P4 14.8 10.9 41.8 67.8 49.1 184.4 174 6 n = 3. P1 – Lolium, P2 – Festuca, P3 – Poa, P4 – Lolium + Festuca + Poa                   217 E.2. Abstract of ANOVA – Field Experiment.  E.2.1. Metal fractionation, total soil metal concentrations and plant metal (root and shoot) concentrations  Table E.6. Cu   Source df Exch. Cu CO3 Cu Oxide Cu Organic Cu Residual Cu Total Cu Root Cu Shoot Cu Treatments (T) 1 * * * * * NS * NS Plants (P) 3 * * * * * * * * Time (Ti) 2 * * * * * * * * P*T 3 * * * NS * * * * T*Ti 2 * NS * * * NS * * P*Ti 6 * * * * * * * * P*T*Ti 6 * NS * * * * * * S.E of mean  1.002 1.66 2.4 2.08 1.24 4.3 13.3 4.43  Table E.7. Pb   Source df Exch. Pb CO3 Pb Oxide Pb Organic Pb Residual Pb Total Pb Root Pb Shoot Pb Treatments (T) 1 * * * NS * * * * Plants (P) 3 * * * * * NS * * Time (Ti) 2 * * * * NS * * * P*T 3 * * * * * * * * T*Ti 2 * * * * * * * * P*Ti 6 * * * * NS * * NS P*T*Ti 6 * * * * NS * * * S.E of mean  0.74 1.79 3.70 4.37 6.79 5.48 6.56 3.20 • n = 3, *F values significant at P<0.05, NS – F values not significant.               218   Table E.8. Mn   Source df Exch. Mn CO3 Mn Oxide Mn Organic Mn Residual Mn Total Mn Root Mn Shoot Mn Treatments (T) 1 NS * * * * NS NS * Plants (P) 3 NS NS * * * * * * Time (Ti) 2 * * * * * * * * P*T 3 * * NS * * * NS * T*Ti 2 NS NS * * * * NS * P*Ti 6 NS NS * * * * * * P*T*Ti 6 NS * NS * * * * * S.E of mean  1.88 0.81 4.11 2.27 3.27 8.23 18.18 73.55  Table E.9. Zn   Source df Exch. Zn CO3 Zn Oxide Zn Organic Zn Residual Zn Total Zn Root Zn Shoot Zn Treatments (T) 1 NS * * NS * NS NS * Plants (P) 3 NS * * * * * * * Time (Ti) 2 * * * * NS * * * P*T 3 * * * * * * * * T*Ti 2 NS * * NS * * * NS P*Ti 6 NS * * * * * * * P*T*Ti 6 * * * * * * * * S.E of mean  5.77 3.63 6.59 8.18 4.67 16.34 8.98 11.6  E.2.2. Bulk Soil Vs Rhizosphere soil, Design – Completely Randomised Factorial Experiment. Metal fractionation and total soil metal concentrations.  Table E 10. Cu   Source df Exch. Cu CO3 Cu Oxide Cu Organic Cu Residual Cu Total Cu Treatments (T) 1 * NS NS * NS NS Plants (P) 3 * * * * * * Soil (S) 1 NS * * * * * P*T 3 * * * * * * T*S 1 NS * * * NS NS P*S 3 * * * * * * P*T*S 3 * * * * * * S.E of mean  1.37 0.42 1.85 2.19 2.17 4.67 • n = 3, *F values significant at P<0.05, NS – F values not significant.  219  Table E.11. Pb   Source df Exch. Pb CO3 Pb Oxide Pb Organic Pb Residual Pb Total Pb Treatments (T) 1 * NS NS NS NS * Plants (P) 3 * * * * * * Soil (S) 1 * * * * * * P*T 3 * * * * * * T*S 1 * NS NS * * * P*S 3 * NS * * * * P*T*S 3 * NS * * * * S.E of mean  0.23 0.69 1.79 3.58 3.96 4.13  Table E.12. Mn   Source df Exch. Mn CO3 Mn Oxide Mn Organic Mn Residual Mn Total Mn Treatments (T) 1 * NS * NS * NS Plants (P) 3 * * * * * * Soil (S) 1 NS * * * * * P*T 3 * * * NS * NS T*S 1 NS * * NS NS NS P*S 3 * * * NS * * P*T*S 3 NS * * NS NS NS S.E of mean  1.42 0.48 2.26 2.96 2.09 14.04  Table E.13. Zn   Source df Exch. Zn CO3 Zn Oxide Zn Organic Zn Residual Zn Total Zn Treatments (T) 1 NS NS * * NS * Plants (P) 3 NS * * * * * Soil (S) 1 * * NS * * * P*T 3 * * * * * * T*S 1 NS NS * * * NS P*S 3 NS * * * * * P*T*S 3 NS * * * NS * S.E of mean  6.67 2.39 5.30 8.18 4.22 11.8  • n = 3, *F values significant at P<0.05, NS – F values not significant.         220 APPENDIX F  List of publications from thesis  Refereed journals  1. Padmavathiamma, P. K. and Li, L. Y. (2007). Phytoremediation Technology: Hyper- accumulation Metals in Plants. Water, Air, and Soil Pollution 184: 105–126.  2. Padmavathiamma, P. K. and Li, L. Y. (2008). Phytoremediation of metal-contaminated soil in temperate humid regions of British Columbia, Canada. International Journal of Phytoremediation 11(6): 575-590.  3. Padmavathiamma, P. K. and Li, L. Y. (2009). Phytoremediation and its Effect on Mobility of Metals in Soil: a Fractionation Study. Land Contamination and Reclamation 17(2), 223-236.  4. Padmavathiamma, P. K. and Li, L. Y. (2009). Phytostabilisation – a sustainable remediation for zinc toxicity in soils. Water, Air, and Soil Pollution 9(3-4), 253-260.  5. Padmavathiamma, P. K. and Li, L. Y. (2009). Effect of amendments on phytoavailability and fractionation of copper and zinc in contaminated soil. International Journal of Phytoremediation. (Accepted: August, 2009).  6. Padmavathiamma, P. K. and Li, L. Y. (2010). Phytoavailability and fractionation of lead and manganese in contaminated soil following application of three amendments. Bioresource Technolgy doi:10.1016/j.biortech.2010.01.149. . Other refereed relevant contributions (e.g., papers in refereed conference proceedings.)  1. Padmavathiamma, P. K. and Li, L. Y. (2006) Metal Hyper-Accumulation in Plants - an Overview of Phytoremediation Technology. Proceedings. 59th Geo-technical Conference. October 1-4.  2. Padmavathiamma, P. K., Li, L. Y. and Lavkulich, L. (2007). Heavy metal contamination and potential of local plants for phytoremediation along Highways. 9th International Conference on Biogeochemistry of Trace Elements (ICOBTE), Beijing, China.  3. Padmavathiamma, P. K. and Li, L. Y. (2007). Comparative phytoremediation potential of five different plant species for Cu in soils of British Columbia. 60th Geo-technical Conference, Ottawa, Canada. October 21-24, 2007.  4. Padmavathiamma, P. K. and Li, L. Y. (2008). Sustainable remediation of Pb along Highways. International Conference on Waste Engineering and Management, CSCE-HKIE, Hong Kong, May 28-30, 2008.   221 5. Padmavathiamma, P. K. and Li, L. Y. (2008). Phytostabilization of metals in Highway soils. GREEN5 International Conference, Vilnius, Lithuania, July 1-4, 2008.  6. Padmavathiamma, P. K. and Li, L. Y. (2008). Cu fractionation and plant accumulation in a soil contaminated with Cu and amended with lime, compost, and organic matter. 61st Canadian Geotechnical Conference, Edmonton. September 21- 24, 2008.  7. Padmavathiamma, P. K. and Li, L. Y Y. (2008). Phytostabilisation– A sustainable remediation for Zn toxicity in soils. International Conference on Environmental Science and Technology (ICEST), Houston, Texas. July 28-31, 2008.       

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.24.1-0069527/manifest

Comment

Related Items